key: cord-351328-ly72scru authors: epstein, jay; burnouf, thierry title: points to consider in the preparation and transfusion of covid‐19 convalescent plasma date: 2020-05-14 journal: vox sang doi: 10.1111/vox.12939 sha: doc_id: 351328 cord_uid: ly72scru this document prepared and endorsed by the working party on global blood safety of the international society of blood transfusion presents elements, as of april 2020, to take into consideration in the preparation and transfusion of covid-19 convalescent plasma as a possible treatment approach of covid-19. the document covers the following important factors to have in mind when considering this treatment: (a) eligibility criteria of convalescent covid-19 patients to donate whole blood or plasma, (b) pre-screening and pre-donation testing of convalescent covid-19 donors; (c) criteria for collection of covid-19 plasma; (d) post-donation treatment of plasma; and (e) it offers recommendations for plasma transfusion. this document provides the perspective as of april 2020 of the working party on global blood safety of the international society of blood transfusion on use of covid-19 convalescent plasma as an experimental treatment for covid-19. the document addresses the following important factors to have in mind when considering this treatment: (a) eligibility criteria of convalescent covid-19 patients to donate whole blood or plasma; (b) pre-screening and pre-donation testing of convalescent covid-19 donors; (c) criteria for collection of covid-19 plasma; (d) post-donation treatment of plasma; and (e) recommendations for plasma transfusion. • because the safety and efficacy of convalescent covid-19 plasma as a treatment for covid-19 are unproven at this time, clinical use of this product should be managed as an experimental therapy consistent with ethical and legal safeguards (informed consent of donors and patients, institutional approval, special labelling as an investigational product, compliance with applicable regulatory requirements). • ideally, covid-19 plasma should be used in the context of an organized research study designed to determine its safety and efficacy in comparison with standard of care or other therapeutic interventions. even if used empirically, it is vital to ensure monitoring of patient outcomes including clinical and laboratory indicators of safety and efficacy to maximize the knowledge that might be gained. • collection and retention of blood specimens from both donors and recipients (pre-and post-treatment) should be performed to permit retrospective determination of the characteristics of an effective product and dosage regimen, and the characteristics of patients most likely to benefit. • general information on the rationale and approach to use of convalescent plasma in virus epidemics can be found in the 'who blood regulators network position paper on use of convalescent plasma, serum or immune globulin concentrates as an element in response to an emerging virus (2017)' [1] . intentional collection of convalescent plasma should be performed only by apheresis in order to avoid unnecessary red cell loss in the donor and to optimize the volume of plasma that can be generated for investigational use. in instances of routine whole blood donation by a previously infected person who meets current suitability criteria, covid-19 convalescent plasma can be prepared by component separation and considered for investigational use if not critically needed for general patient care. transfusion of whole blood to provide convalescent plasma should be avoided unless use of whole blood is clinically indicated. (d) volume of plasma to be collected: at least 200-600 ml (without anticoagulant) based on the procedure and regulatory limits. (e) plasma units intended for use as convalescent plasma should be clearly labelled (isbt128 product description codes for convalescent plasma are available for establishments using the isbt128 information standard). (f) the first plasma donation can be followed by further donations at a frequency compliant with local regulations and taking into full account the health status of the donor including monitoring of serum protein levels. in many jurisdictions, the interval between apheresis plasma donations of 600 ml or more should not be less than 7 days and that between whole blood donations should be at least 8 weeks. (d) post-donation treatment of plasma: (a) where feasible, pathogen inactivation of plasma using a licensed technology is highly desirable to control residual risks of transfusion-transmitted infectious diseases and to allay concern about possible superinfections with sars-cov-2. who blood regulators network: position paper on use of convalescent plasma, serum or immune globulin concentrates as an element in response to an emerging virus who blood regulators network (brn): donor selection in case of pandemic situations update on transfusion-related acute lung injury this document was endorsed by the organizing committee of the working party on global blood safety of the disclaimer jay epstein's contributions to this article reflect his own views and should not be construed to represent fda's views or policies. key: cord-323656-bzefn894 authors: yoo, jin-hong title: convalescent plasma therapy for corona virus disease 2019: a long way to go but worth trying date: 2020-04-06 journal: j korean med sci doi: 10.3346/jkms.2020.35.e150 sha: doc_id: 323656 cord_uid: bzefn894 nan dosage and administration protocols have not been standardized yet. in both cases, plasma was administered when antiviral drugs and steroids were given. it is hard to tell that the successful treatment is not necessarily due to plasma, and it cannot be refuted even if it is interpreted as an effect of antiviral agent or steroid. or it is possible that these three elements were combined to create a synergistic effect. but i'm going to change the way of interpretation. given the mechanism of convalescent plasma therapy, i think this combination is rather worth being recommended. the targets of covid-19 treatment should be largely divided into two categories. first, it is aimed at the virus itself. the first thing you can think of is destroying the body of the virus. however, destroying the virus itself is a concept of disinfection and is too dangerous for humans to apply. as a therapeutic agent, there are drugs that inhibit rna-dependent rna polymerase by inhibiting the replication of viruses (e.g., remdesivir), or drugs that inhibit protease (e.g., lopinavir/ritonavir). 1,13 another target is angiotensin converting enzyme 2 (ace2), a gatekeeper and receptor for viruses to enter human cells. by raising the intracellular ph, glycosylation of ace2 can be prevented to block the entry of the virus (e.g., chloroquine), 13,14 or it can be prevented from binding to ace2 in advance by sticking to the spike protein of the virus. 15, 16 the latter, not the former, is the antibody. considering the above treatment mechanisms, it can be seen that it is difficult to succeed with only one mechanism to treat covid-19. blocking a virus with antibodies is not enough to win the battle. we must also suppress the replication of the virus, and prepare for a cytokine storm that occurs during treatment. 17 in conclusion, it makes no sense as to which of these treatment methods was a decisive factor in the successful treatment. rather, it is necessary to combine all of these to engage in treatment. we need to examine another important problem in plasma treatment. plasma therapy itself has important complications. examples are transfusion-related acute lung injury (trali), circulatory overload, or anaphylaxis. 18 fortunately, no adverse events have been reported. nevertheless, these complications should always be a concern. there is also the possibility of side effects that have been raised recently. it is the antibodydependent enhancement of entry (ade). neutralizing antibodies, once bound to the spike protein of the virus, cause a conformational change of the spike and, consequently, could trigger the paradoxical result of better entry into human cells through the igfc receptor. 19 -21 this side effect has not yet been realized, but should be kept in mind in the future of plasma treatment and vaccine development. convalescent plasma therapy gives us a lot of hope, but there are challenges to overcome. in the implementation, thorough ethical verification is required, and donor selection criteria should be strictly enforced. and it needs further extensive research to see if it really works. to this end, i think that institutional support is required to approve every attempt as quickly as possible. again, it is time to focus all of our capabilities on treatment. drug treatment options for the 2019-new coronavirus (2019-ncov) convalescent plasma: new evidence for an old therapeutic tool? convalescent plasma as a potential therapy for covid-19 use of convalescent plasma therapy in two covid-19 patients with ards in korea treatment of 5 critically ill patients with covid-19 with convalescent plasma the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis use of convalescent plasma therapy in sars patients in hong kong experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a taiwan hospital treatment with convalescent plasma for influenza a (h5n1) infection convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection use of convalescent whole blood or plasma collected from patients recovered from ebola virus disease for transfusion, as an empirical treatment during outbreaks viral load kinetics of sars-cov-2 infection in first two patients in korea remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro chloroquine is a potent inhibitor of sars coronavirus infection and spread perspectives on monoclonal antibody therapy as potential therapeutic intervention for coronavirus disease-19 (covid-19) potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology adverse effects of plasma transfusion molecular mechanism for antibody-dependent enhancement of coronavirus entry anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a ph-and cysteine proteaseindependent fcγr pathway antibody-dependent sars coronavirus infection is mediated by antibodies against spike proteins key: cord-271764-um001ffd authors: garraud, olivier title: passive immunotherapy with convalescent plasma against covid-19? what about the evidence base and clinical trials? date: 2020-06-27 journal: transfus apher sci doi: 10.1016/j.transci.2020.102858 sha: doc_id: 271764 cord_uid: um001ffd nan neutralizing antibodies-after clinical recovery of the donor? and for how long (in other words, is the serological neutralizing response long-lasting)? are titers of neutralizing antibodies in a recovering person high enough to enable protection in a recipient (or is there a need for preparing so-called hyperimmune immunoglobulins)? next, questions arise relative to donors: can convalescent persons safely give plasma (in relatively high volume, representing an extracorporeal circulation and exposure, to non-negligible levels of calcium citrate)? do those persons come forwardvoluntarily and freely, in the absence of pressure, or were there external motivations such as incentives or remuneration [16] ? last, questions arise regarding testing and qualification of donated plasma:will they be derogation of the normal rules or will restrictive qualification be applied to all donations at the expense of a potential wastage of plasma presenting an ad hoc level of antibodies but without the standard safety guarantee? derogations could concern e.g. doubtful seropositivity of other viral markers; high-titer anti-hla antibodies; irregular anti-red blood cell antibodies; history of transfusion, a contraindication e.g. in france and some other countries; etc. next come the scientific questions relative to the timeframe for transfusing convalescent plasma to recipients. at which phase of the disease to transfuse the immune plasma to best neutralize the viral replication in order to prevent tissue/organ complications? indeed, it would make sense that "the earlier the best"; this would indeed require smaller volumes of plasma. however, how to decipher between infected persons at-risk of manifesting complications in whom plasma therapy is sound, versus the many others who will not present with complications, in whom the exposure to plasma could cause an unnecessary risk and no or little benefit? convalescent plasma is by all means a rare resource and only its judicious application for treatment will be acceptable, not to waste the resource. then, which protocol to apply to ensure that there is enough contact between neutralizing antibodies and the virus (and at which site)? in all, what would be the best protocols, first to collect, secondly to qualify, and thirdly to apply specific plasma? dozens of hopes and disappointments characterize covid-19 therapeutic options, in particular because there have to quickly been communications about uncontrolled or methodologically unsound trials. this is explained first by the race against time in this devastating pandemic; then because even renowned journals fast-track publications prior to peer-review, often against publication ethics; and, last,because of the media hunt for spectacular discoveries, and also the identification of moguls as novel show-persons who surrogate the usual tv-stars. it is obvious that ethically sound, quality reviewed, well conducted clinical trials are essential to situate convalescent plasma therapy among the therapeutic arsenal to treat sars-cov-2 infection. controls would also be essential, ie against non-convalescent plasma or non-specific j o u r n a l p r e -p r o o f immunoglobulins; the situation will be complexified at a time when even standard intravenous immunoglobulins (ivig) have been proposed in certain severe situations, especially with the aim to dampen the inflammatory phase in severe presentations (further, current ivig forms seem to have certain antiviral properties [17] ). indeed, since the discovery of numerous thrombotic complications among the severe presentations of covid-19 [18] [19] [20] , it cannot be excluded that normal plasma factors resolve the dic-like symptomatology or counteract the effects of lupus-like antibodies, or sooth the blood vessel endothelium if the disease associates to endotheliopathy (as was seen relative to ebola-virus infection and convalescent plasma therapy [21] ). further, what about the ethics of transfusing supposedly large volumes of non-specific plasma as a control to a person in danger of developing severe complications of sars-cov-2? last, the case of hemovigilance of plasma therapy must be addressed, to not harm recipients, especially having in mind that one of the most frequent hazards of transfusion is lung injury; it would be inappropriate to superimpose this hazard on the pulmonary complication of covid-19. of major interest is one of the first trials published so farconcerning about 5,000 recipients-that has identified only limited and non-unexpected transfusion complications [22] . in aggregate, convalescent plasma therapy as a rescue treatment is sound considering a hundred years of experience [1] , when no obvious form of treatment has yet been made available and in the absence of soon-coming vaccine. however, this raises a flurry of ethical questions that each need to be properly addressed. the question of alleviating ethical clearance for clinical trials has been raised by many investigation centers relative to covid-19, considering the urgency in obtaining encouraging data; this is vigorously debated, however, to not raise false hopes nor to complicate the proper clinical management of patients [23, 24] . in our opinion, it is unfortunate that the situation has not been anticipated, as recent sars-cov-1 and sars-mers-and also ebola-virus-infections raised similar questions; the controlled use of plasma therapy on a large scale has not been possible in the preceding situations as the option was made available too late, at a time where the spreading epidemic reversed to sporadic cases. it would be urged that there is a general preparedness plan to evaluate the assets and liabilities of convalescent plasma to treat emergent infections; as a matter of fact, plasma therapy of convalescent plasma has not been thought of in a recent position paper on preparedness plans [25] . models should be prepared (perhaps in animal hosts) and ethical questions should be prepared ahead of an exception context, such as the present one when elementary liberties have been notched despite the existence of democratic regimens in most industrialized countries. clinical trials to evaluate the quality, the efficacy and the safety of convalescent plasma therapy as soon as possible after the onset of an epidemic threat would be mostly valuable. plasma therapy against infectious pathogens, as of yesterday, today and tomorrow convalescent plasma in covid-19: possible mechanisms of action convalescent plasma therapy for covid-19: state of the art effect of convalescent plasma therapy on viral shedding and survival in covid-19 patients treatment of 5 critically ill patients with covid-19 with convalescent plasma effectiveness of convalescent plasma therapy in severe covid-19 patients testing an old therapy against a new disease: convalescent plasma for covid-19 collecting and evaluating convalescent plasma for covid-19 treatment: why and how? vox sang 2020, ahead of print treatment for emerging viruses: convalescent plasma and covid-19 the convalescent sera option for containing covid-19 convalescent plasma to treat covid-19: possibilities and challenges points to consider in the preparation and transfusion of covid-19 convalescent plasma convalescent plasma to treat coronavirus disease 2019 (covid-19): considerations for clinical trial design covid-19 convalescent plasma: phase 2. transfusion 2020 ahead of print may 6 current studies of convalescent plasma therapy for covid-19 may underestimate risk of antibody-dependent enhancement plasma from donors convalescent from sars-cov-2 infection -a matter of priorities currently available intravenous immunoglobulin contains antibodies reacting against severe acute respiratory syndrome coronavirus 2 antigens lupus anticoagulant and abnormal coagulation tests in patients with covid-19 coagulopathy and antiphospholipi antibodied in patients with covid-19 immune thrombocytopenic purpura in a patient with covid-10 early safety indicators of covid-19 convalescent plasma in 5,000 patients clinical trials and the covid-19 pandemic covid-19 and moral imperialism in multinational novel coronavirus and old lessons -preparing the health system for the pandemic acknowledgement: the author would like to express his gratitude to prof. gail rock for her fruitful advice and help in editing this manuscript.j o u r n a l p r e -p r o o f key: cord-282252-07uzz649 authors: sahu, kamal kant; jindal, vishal; siddiqui, ahmad daniyal; cerny, jan; gerber, jonathan m. title: convalescent plasma therapy: a passive therapy for an aggressive covid‐19 date: 2020-05-21 journal: j med virol doi: 10.1002/jmv.26047 sha: doc_id: 282252 cord_uid: 07uzz649 as of may 19, 2020, there are in total 4,986,200 laboratory‐confirmed coronavirus disease‐2019 (covid‐19) cases. 2% (45,425) out of 2,657,390 active covid‐19 cases are critically ill and might be requiring intensive care support.(1,2) unfortunately, even after six months since its first detection, we still do not have any definitive treatment options for covid‐19 pneumonia. this article is protected by copyright. all rights reserved. author contribution statement: all authors have seen the manuscript and agree to the content and data. all the authors played a significant role in the paper the basic concept for use of convalescent plasma in covid-19 is as a delivery system for viral neutralizing antibodies, that is to confer passive immunity. given the fact that we do not have reliable targeted drugs or a vaccine yet, the option of convalescent plasma seems reasonable to boost the immune system of infected patients or susceptible population immediately. this is not a new concept, rather this has been utilized for over 100 years, even predating the discovery of antibiotics. with regards to the previous outbreaks, experience with convalescent plasma have shown mixed results(4-6). soo et al reported a low mortality rate (p =0.049) and shorter hospital stay (p= 0.001) in patients with sars by using convalescent plasma (4) . contrarily, the use of convalescent plasma therapy has been found to be of uncertain benefit in the 2013 african ebola epidemic (6) . this article is protected by copyright. all rights reserved. many studies have confirmed that not all ebola survivors have anti-ebola antibodies and hence plasma extraction from such donors might not be beneficial for the treatment of ebola disease (7) . preliminary results on using convalescent plasma in covid-19 patients have shown positive results (8, 9) . shen patients (10, 11) the major findings of this review were [1] reduced mortality in critically ill patients [2] disappearance of sars-cov-2 rna was observed in the majority of patients [3] improvement in clinical symptoms and radiological shadows of the patients after convalescent plasma therapy [4] no significant adverse effects secondary to plasma therapy were noted. although the convalescent plasma therapy concept is old, the covid-19 disease is just six months old. hence, a large degree of uncertainty exists as to donor selection, patient eligibility, indications, and side effects that merit further discussion (11) . this article is protected by copyright. all rights reserved. should have completely resolved at least 14 days before donation and negative covid-19 pcr from nasopharyngeal swab. once it is confirmed that the proposed donor is no longer contagious, the next step would be to see if the donor has sufficient antibody levels to donate? this can be done by measuring sars-cov-2 antibody levels to ensure sufficient titers in the donor's circulation. the fda recommends a sars-cov-2 neutralizing antibody titer of at least 1:160 as an inclusion criterion for donor selection. if such a matched unit is not available, the fda suggests that a titer of 1:80 may be considered acceptable. convalescent plasma therapy involves many logistical challenges, including the donor's availability and willingness; apheresis center capacity; storage and transportation of plasma concentrate; and testing for the adequacy of antibody titers. considering the aforementioned limitations and the potential risks, appropriate triage systems should be utilized; hence, plasma therapy use is currently restricted only to critically ill patients. this article is protected by copyright. all rights reserved. the fda recommends two clinical indications for the current usage of convalescent plasma therapy in covid-19 patients(12) scenario a (severe disease) which is defined as one or more of the following: dyspnea, rr ≥ 30/min, blood oxygen saturation ≤ 93%, pao2 / fio2 ratio < 300, and radiological worsening with the appearance of lung infiltrates > 50% within 24 to 48 hours. it is unknown how long such protection might last but based on the amount and type of transfused antibody, immunity could last from weeks to months. another important factor is the timing of plasma therapy infusion. as viremia is expected to be maximum in the first week, the early infusion is likely to give the best response. possible mechanisms are viral neutralization and antibody-dependent cellular cytotoxicity and/or phagocytosis. this helps in not only clearing the viremia but also could potentially eradicate the reservoir of infected host cells. currently, the only antibody source available for urgent use is from convalescent plasma from recovered patients. it is anticipated that as more people contract and recover from covid-19, the number of potential donors will rise. this article is protected by copyright. all rights reserved. on april 3, 2020, the fda cleared the path for the use of this potential lifesaving therapy under any of the following three routes[1] enrollment in a clinical trial, [2] via the national expanded access treatment protocol, and [3] under a single patient emergency investigational new drug application (eind). armed with fda approval, researchers are now in the process of conducting placebo-controlled trials to test convalescent plasma, at numerous hospitals, including johns hopkins, the mayo clinic (nct04325672), and washington university in st. louis (table 1) . lacking a vaccine and with limited antiviral options against sars-cov-2, this is an ideal time to try convalescent plasma therapy in covid-19 patients. preliminary results are very encouraging, and none of the studies have thus far shown any significant adverse reactions. however, as experience is still limited, vigilance for potential side effects of convalescent plasma therapy is advised. as with any other blood product transfusion, there are certain common, predictable, or known side effects that also apply to convalescent plasma therapy, such as transfusion-related infections, serum sickness, fluid overload, and transfusion-related acute lung injury. by following diligent modern blood banking techniques and transfusion precautions, the incidence of these unwanted events can be minimized at any center; and the cumulative risk of any lifethreatening reactions is < 1%. although a theoretical risk that of antibody-dependent enhancement of infection, this has not been witnessed to date.; and there are no data thus far to suggest any increased risk of convalescent plasma over ordinary fresh frozen plasma (13) . this article is protected by copyright. all rights reserved. while awaiting an effective vaccine and/or antiviral agent for covid-19, experimental therapies are currently being testing in clinical trials (14) . plasma therapy has so far provided encouraging outcomes, without any serious events. we anticipate an upsurge in the use of convalescent plasma over the next several months, for the treatment of severely ill patients and perhaps with a role earlier in the course of illness and/or for prophylaxis. the american red cross, the u.s. government, investigators at mayo clinic (www.uscovidplasma.org), and many others across the country are now hard at work identifying appropriate donors and establishing testing to confirm neutralizing antibodies in a timely fashion. as antibody testing is validated, it should help guide the more effective use of convalescent plasma. in addition, efforts are underway to pursue production of a covid-19 immune globulin, which might provide a more reliable, more effective, and more readily available plasma-based therapy against this formidable virus. latest updates on covid-2019: a changing paradigm shift covid-2019: update on epidemiology, disease spread and management the convalescent sera option for containing covid-19 retrospective comparison of convalescent plasma with continuing high-dose methylprednisolone treatment in sars patients use of convalescent plasma therapy in sars patients in hong kong evaluation of convalescent plasma for ebola virus disease in guinea anti-ebola virus antibody levels in convalescent plasma and viral load after plasma infusion in patients with ebola virus disease treatment of 5 critically ill patients with covid-19 with convalescent plasma effectiveness of convalescent plasma therapy in severe covid-19 patients convalescent plasma transfusion for the treatment of covid-19: systematic review. journal of medical virology treatment with convalescent plasma for covid-19 patients in wuhan 12. recommendations for investigational covid-19 convalescent plasma | fda molecular mechanism for antibody-dependent enhancement of coronavirus entry current perspective on pandemic of covid-19 in the united states key: cord-261653-0vtghtp7 authors: andersen, kylie j.; klassen, stephen a.; larson, kathryn f.; ripoll, juan g.; senefeld, jonathon w.; clayburn, andrew j.; shepherd, john r.a.; tseng, andrew s.; wiggins, chad c.; johnson, christopher p.; miller, andrew d.; baker, sarah e.; wright, r. scott; winters, jeffrey l.; stubbs, james r.; joyner, michael j.; van buskirk, camille m. title: recruitment strategy for potential covid-19 convalescent plasma donors date: 2020-09-21 journal: mayo clin proc doi: 10.1016/j.mayocp.2020.09.021 sha: doc_id: 261653 cord_uid: 0vtghtp7 nan coronavirus disease 2019 (covid-19) represents a pandemic and global health crisis. in the first wave of the disease in the us, covid-19 was diagnosed in approximately two million individuals and contributed to over one-hundred thousand deaths. 1 the number of covid-19 diagnoses and related deaths are anticipated to perpetuate in subsequent waves of the disease. 2 the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) which causes covid-19 appears to be a new human pathogen with limited efficacious treatments including the antiviral, remdesivir and the glucocorticoid, dexamethasone. 3, 4 however, there is robust historical precedent to anticipate that human convalescent plasma is a viable option for mitigation and treatment of covid-19. 5, 6 human convalescent plasma leverages the antibody response of a recently-sick and recovered covid-19 patient as a plasma donor. 7 the donated plasma which is rich in neutralizing antibodies to the sars-cov-2 virus is transfused to a currently-sick covid-19 patient to mitigate on-going symptomology and induce passive immunity. 7 thus, the us food and drug administration (us fda) in collaboration with the mayo clinic and national blood banking community developed a national expanded access program (eap) to collect and distribute convalescent plasma donated by covid-19 survivors. 8 theoretically, within four to six weeks of the onset of the us covid-19 outbreak, human convalescent plasma should have been readily available due to the existing plasma collection infrastructure within the blood banking community 9 and the large numbers of covid-19 survivors who could have donated high-titer immunoglobulin-containing plasma. 10 however, logistical issues produced a convalescent plasma fulfillment gap that created an urgent need to develop strategies to recruit eligible donors at national, regional, and local blood centers. thus, the present narrative overviews the strategy developed by our team to identify and recruit covid-19 survivors to donate convalescent plasma at the mayo clinic blood donor center in rochester, minnesota. emerging data from the covid-19 pandemic support the use of convalescent plasma therapy. there is evidence to suggest that sars-cov-2 elicits a robust immune response with high levels of antibodies and immunoglobulins (m and g) between 11-21 days after the onset of covid-19, suggesting a relatively large window of time and high probability of successful extraction of high-titer anti-sars-cov-2 plasma from donors. 10 consistent with this observation, convalescent plasma treated covid-19 patients had large reductions in serum sars-cov-2 j o u r n a l p r e -p r o o f serum viral loads after convalescent plasma infusion, suggesting a viral neutralizing effect of the anti-sars-cov-2 antibodies. 11 some theoretical concerns for the use of convalescent plasma therapy for covid-19 have been raised, including the risk of transfusion-related lung injury (trali), transfusion-related circulatory overload (taco), and antibody-dependent enhancement of covid-19. 12 however, in a large study of 20,000 patients transfused with convalescent plasma there was no signal of toxicity beyond what is expected from plasma use in severely-ill patients. 8, 13 while the efficacy of convalescent plasma remains unknown, preliminary investigations have provided a signal of possible benefit in severely-ill covid-19 patients. 14, 15 thus, recruitment of covid-19 survivors was fundamental to meet the rapidlygrowing demand for convalescent plasma as the pandemic progressed and clinical interest in convalescent plasma therapy increased. rochester, minnesota required a strategy to interface with the community of recovering covid-19 patients and recruit eligible convalescent plasma donors. to accommodate the growing magnitude and geographical spread of covid-19 survivors and the changing convalescent donor eligibility criteria outlined by regulatory agencies and local institutions, we required this strategy to be inherently modifiable. overall, this recruitment strategy utilized a simple survey, an algorithm for triaging donors, a workflow for connecting donors with mayo clinic blood donor center, a team of physician navigators (including medical students) to screen eligible donors, and a support center for donor questions. this strategy may be adopted by other institutions to rapidly increase convalescent plasma donor recruitment. to identify previously-ill recovered covid-19 survivors that satisfied present and changing eligibility criteria to donate convalescent plasma, we employed a brief online survey hosted by the research electronic data capture (redcap) system. 16, 17 to reduce user time-burden this survey was limited only to questions deemed necessary to determine donor eligibility and to maximize the retention of interested volunteers ( table 1) respondents were deemed ineligible to donate convalescent plasma and were immediately excluded from follow up by our team if they i) reported no positive covid-19 laboratory test, ii) submitted an incomplete survey, iii) did not authorize follow up contact by our team, iv) or could not be contacted by our team for follow-up (figure 1) . volunteers were also excluded from follow up by our team if they declined contact authorization at any time. ineligible respondents received an automated email to communicate both our gratitude and the reason for their j o u r n a l p r e -p r o o f ineligibility. this automated email also included a new call to action for interested volunteerswe encouraged the individuals (if eligible) to perform a standard blood donation to prevent future blood shortages and direct them to the aabb website (http://covidplasma.org/) which provides blood donation information. also, our donor service center encouraged interested potential donors who reported covid-19 symptoms but did not receive a covid-19 clinical test to contact their healthcare provider for a serology test and then fill out the recruitment survey again if they were sero-positive. our web-based recruitment survey and all e-mail communications to interested potential donors contained the e-mail address for our convalescent plasma service center. the service center team used available resources from the us fda, mayo clinic, and the blood banking community to support questions regarding donor eligibility and covid-19 testing. 9,18 also, the service center supported the physician navigator team by providing ongoing communication with screened eligible donors. this convalescent plasma donor recruitment strategy had several key strengths: 1) the recruitment strategy had inherent scalability to meet the growing magnitude of recovering covid-19 patients and enabled filtering by donor location to link the volunteer to a convenient donation center. 2) this strategy reduced the administrative and clinical workload associated with screening potential convalescent plasma donors experienced by transfusion staff at the mayo clinic blood donor center. thus, the blood donation team was able to continue collecting apheresis platelet, platelets, and whole blood donations while also scaling up convalescent plasma donations during the ongoing pandemic. 3) the simple web-based screening survey optimized volunteer convenience and the screening algorithm enabled rapid identification of currently eligible volunteers, retained interested volunteers that were not eligible upon initial survey completion, and provided a new call to action for those that did not meet convalescent plasma donation eligibility criteria -perform a standard blood donation, if eligible. our strategy for interfacing with the community of covid-19 survivors may be expanded through the following avenues: 1) by introducing plasma fractionators into our recruitment network, we may recruit donors to contribute to a hyperimmune igg product for covid-19. two important populations of eligible donors would be immediate candidates for the plasma fractionation route: i) individuals that have antibody titers below the threshold thought to be efficacious for covid-19 treatment and ii) volunteers ineligible for standard blood donation, such as multiparous women with human leukocyte antigen antibodies. 2) there is the opportunity to leverage electronic medical records to, upon a positive covid-19 test result, automatically alert the ordering physician to provide the patient with the link to our donor recruitment survey and support center email address. as the availability of serological tests increases, this avenue will enhance the volume of donors that did not receive an initial diagnostic test. survivors. based on the responses to three key questions within the convalescent plasma donor recruitment survey -regarding covid-19 laboratory test result, covid-19 symptomology, and current residence -our team delineated interested volunteers into four categories: i) immediately eligible, ii) soon to be eligible, iii) eligible for other efforts, and iv) ineligible. regardless of the categorization, all respondents authorizing follow-up interactions were contacted by our team of physician navigators or an automatically generated email to engage in additional covid-19 related donor screening or to initiate a new call to action for blood or convalescent plasma donation. expanded details are available in donor recruitment strategy overview. j o u r n a l p r e -p r o o f usa facts. coronavirus locations: covid-19 map by county and state remdesivir for the treatment of covid-19-preliminary report dexamethasone in hospitalized patients with covid-19-preliminary report meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? return to the past: the case for antibody-based therapies in infectious diseases the convalescent sera option for containing covid-19 convalescent plasma in 5,000 patients deployment of convalescent plasma for the prevention and treatment of covid-19 antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 treatment of 5 critically ill patients with covid-19 with convalescent plasma covid-19 convalescent plasma: now is the time for better science safety update: covid-19 convalescent plasma in 20,000 hospitalized patients effect of convalescent plasma therapy on time to clinical improvement in patients with severe and life-threatening covid-19: a randomized clinical trial convalescent plasma treatment of severe covid-19: a matched control study. medrxiv research electronic data capture (redcap)-a metadata-driven methodology and workflow process for providing translational research informatics support the redcap consortium: building an international community of software platform partners convalescent plasma key: cord-280221-s6oxq772 authors: montelongo-jauregui, daniel; vila, taissa; sultan, ahmed s.; jabra-rizk, mary ann title: convalescent serum therapy for covid-19: a 19th century remedy for a 21st century disease date: 2020-08-12 journal: plos pathog doi: 10.1371/journal.ppat.1008735 sha: doc_id: 280221 cord_uid: s6oxq772 nan global recognition and revolutionized the way infectious diseases were treated, and in 1901, emil von behring was awarded the nobel prize for medicine for his work, which served as a basis for treatment of multiple diseases in the 1900s as well as the development of vaccines [15] . in fact, there are numerous examples throughout history in which convalescent serum was used with some degree of success to treat an array of diseases, including rheumatic fever [16] , scarlet fever [17] , mumps [18] , measles [18, 19] , chickenpox [18] , and pneumococcal and meningococcal infections [20] (fig 1) . most notable use was during the spanish flu pandemic (1918 to 1920) , where meta-analysis studies showed a significantly reduced mortality risk in patients treated with convalescent serum [8, 12] . however, with the advent of antimicrobials, by the middle of the 20th century, the use of serum therapy had declined. nevertheless, the interest in passive antibody therapy has been renewed periodically when new epidemics or pandemics have emerged. one example is during the ebola virus (ebov) outbreak in 1976 in the democratic republic of congo, where an infected laboratory worker recovered after transfusion with convalescent plasma containing anti-ebov antibodies. similarly, in 1979, patients with argentine hemorrhagic fever virus treated with convalescent plasma had a lower mortality rate compared with subjects treated with normal plasma, and similar results were reported for subsequent epidemics of the disease [21] . over the following decades, convalescent plasma therapy was successfully employed during the h1n1 swine influenza pandemic (2009), the h5n1 avian flu epidemic (2003), as well as during the ebov outbreak in west africa in 2013. most relevant and encouraging is the use of convalescent plasma during 2 previous coronavirus epidemics: severe acute respiratory syndrome (sars) in 2003, and middle east respiratory syndrome (mers) in 2012 [21] . the high degree of success in achieving favorable clinical outcomes during these coronaviruses outbreaks establishes a strong precedent and supports the notion that convalescent plasma could be a viable option for treatment of covid-19 patients, particularly upon early administration [6, 9, 12, [21] [22] [23] . the convalescent plasma therapeutic approach is based on the principle of passive antibody therapy, a short-term strategy whereby antibodies from the blood of someone who recovered from an infection can be administered to protect or treat another person [6, 21] . effectively, the end goal is the same as vaccines, making antibodies against a specific infectious agent readily available. for instance, a vaccine relies on the host immune cells (b lymphocytes specifically) to produce antibodies after antigen recognition and signal amplification by the immune system, a process that may take weeks [24] ; on the other hand, in the case of passive antibody therapy, the process is expedited by providing a patient with immediate immunity when the premade antibodies are given. therefore, for covid-19 patients, the expedited approach could prove lifesaving. nevertheless, this advantage does not come without caveats, as immunization with passive antibody therapy is typically of shorter-term protection, in part because of the half-life of antibodies in circulation [25] and lack of new production by b lymphocytes. today, passive antibody therapy relies primarily on pooled immunoglobulin preparations that contain high concentrations of antibodies. in contrast, plasma has been used emergently in epidemics in which there is insufficient time or resources to generate immunoglobulin preparations [21] . despite the high rate of sars-cov-2 infection, the relatively low mortality rate provides a rich pool of donors [26] . however, potential covid-19 donors must meet several eligibility criteria that ensure the donor has antibodies against sars-cov-2 and lacks the presence of other types of infections [21, 27] . additionally, only plasma with high anti-sars-cov-2 titers of immunoglobulins g and m (igg and igm) are used. once collected, plasma can be tested and administered within hours, following conventional donor-patient blood compatibility typing [27] . in addition to rapid mobilization, this therapeutic approach is also versatile in applicability as it can be used for prophylaxis or treatment, as illustrated in fig 2. in the case of prophylaxis, a subject considered at high risk for infection (because of age or underlying medical conditions or who is likely to be in contact with people with covid-19) could be administered convalescent plasma or neutralizing antibodies for protection against infection. alternatively, plasma can be administered to treat subjects who have contracted the infection but have not made sufficient antibodies against the virus yet in order to augment their immune response, improve disease course, and enhance recovery [21] . however, it is important to note that passive antibody therapy is most effective when administered prophylactically or implemented early after the onset of symptoms [21] . convalescent plasma with neutralizing antibodies is currently being used for investigational purposes in the covid-19 pandemic, and preliminary results from 2 small studies performed in china are encouraging. a pilot study exploring the feasibility of convalescent plasma transfusion to rescue a group of 10 patients with severe disease showed that 1 dose (200 ml) of convalescent plasma with high neutralizing antibody titers was well tolerated, resulted in disappearance of viremia, and improved clinical symptoms in all patients within days of administration [8] . similar results were reported from another study with 5 critically ill patients on mechanical ventilation [7] . although these small, nonrandomized studies had limitations, these findings indicate that convalescent plasma could be a promising rescue option for severe covid-19 [21] . although convalescent plasma therapy is considered a relatively safe therapeutic modality, there are some potential risks [12] . one theoretical complication that may arise is an antibodymediated proinflammatory disease enhancement known as antibody-dependent enhancement (ade), whereby antibodies that developed during a prior infection exacerbate severity of the disease [12, 28] . the transfer of these antibodies may aberrantly activate fragment crystallizable (fc) or complement receptors, increasing recruitment of proinflammatory cytokines and chemokines to the site of infection and causing severe tissue damage [29] [30] [31] . additionally, the presence of non-neutralizing antibodies may exacerbate viral endocytosis or phagocytosis into host cells via fc receptors, potentializing viral replication [29, 32] . however, although this phenomenon is well known with dengue and other viral diseases, there have not been any reported ade cases with the use of convalescent plasma for sars, mers, or covid-19 [12, 29, [32] [33] [34] [35] . nevertheless, it is crucial to have a clear understanding of the role of the recipients' immune response [12] . transmission of the virus through transfusion is another concern; however, the risk is relatively low because of strict transfusion protocols, and when used for treatment purposes, the recipient is already infected [12, 36] . the success of convalescent plasma therapy hinges on the availability of plasma with high concentrations of antibodies, which may not be a major limitation for covid-19 because sars-cov-2 has been shown to elicit high neutralizing antibody titers in recently convalesced individuals [7] [8] [9] . apheresis is an automated technology that allows for selective collection of a blood fraction while other components can be transfused back to the donor. therefore, for donation of convalescent plasma, plasmapheresis is recommended because it is highly efficient and approximately 400-800 ml of plasma can be collected in a single donation, providing 2-4 units of convalescent plasma for transfusion [21] . there are some practical and logistical limitations for the implementation of a large-scale convalescent plasma transfusion program such as training of study personnel, recruitment of donors, and transport of plasma to hospitals, as well considerations for plasma shelf-life and half-life of antibodies in the plasma [21, 36] . although large-scale randomized clinical trials will ultimately confirm the safety and efficacy of convalescent plasma therapy for covid-19, a recent safety study by joyner and colleagues [12] provided encouraging data. by analyzing key safety metrics following transfusion of convalescent plasma in 5,000 hospitalized adults with severe or life-threatening covid-19, the incidence of serious adverse events was found to be <1%, and the 7-day mortality incidence was 14.9%. these early indicators are encouraging and highly supportive of the use of convalescent plasma as a rescue therapy in hospitalized patients, given that the reported fatality rate of covid-19 among patients admitted to the intensive care units (icus) is 57% [37] . in fact, the low risk indicated by the study support expanding the use of convalescent plasma therapy in less ill individuals [6] . the global reach of the covid-19 pandemic and the desperate need for effective treatments have provided an impetus to develop convalescent plasma therapy into a viable (albeit shortterm) treatment option particularly for the critically ill. although its efficacy and safety have not yet been fully proven, convalescent plasma therapy for covid-19 patients is projected to be a safe and potentially effective therapy for prophylaxis and treatment. however, it is critically important to perform rigorous randomized controlled trials to confirm efficacy and safety and to provide evidence for improved meaningful clinical outcomes. nevertheless, despite the nuanced challenges, the substantial evidence of benefit with use for prior viral infections offers strong precedent for convalescent plasma as a therapeutic approach. importantly, efforts should be focused not only on evaluation of the feasibility of plasma treatment for infectious diseases but also ensure that use of convalescent plasma therapy takes place according to ethical and controlled conditions. genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding the sars, mers and novel coronavirus (covid-19) epidemics, the newest and biggest global health threats: what lessons have we learned? origin and evolution of pathogenic coronaviruses bat coronaviruses in china remdesivir for the treatment of covid-19-preliminary report the convalescent sera option for containing covid-19 treatment of 5 critically ill patients with covid-19 with convalescent plasma effectiveness of convalescent plasma therapy in severe covid-19 patients anti-sars-cov-2 virus antibody levels in convalescent plasma of six donors who have recovered from covid-19 treatment with convalescent plasma for covid-19 patients in wuhan convalescent serum lines up as first-choice treatment for coronavirus early safety indicators of covid-19 convalescent plasma in 5,000 patients ueber das zustandekommen der diphtherie-immunitä t und der tetanus-immunitä t bei thieren untersuchungen über das zustandekommen der diphtherie-immunitä t bei thiere. philipps-universitä t marburg remembering emil von behring: from tetanus treatment to antibody cooperation with phagocytes the action of vaccines and of concentrated antistreptococcus serum in experimental streptococcal arthritis serum treatment of scarlet fever university of nebraska the use of convalescent serum in the treatment of measles, chickenpox, mumps and whooping cough, including the prophylactic value of parental blood treatment of preeruptive measles with convalescent serum serum therapy revisited: animal models of infection and development of passive antibody therapy deployment of convalescent plasma for the prevention and treatment of covid-19 convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection hyperimmune iv immunoglobulin treatment: a multicenter double-blind randomized controlled trial for patients with severe 2009 influenza a (h1n1) infection principles of vaccination public health foundation. 13th therapeutic antibodies: successes, limitations and hopes for the future severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges points to consider in the preparation and transfusion of covid-19 convalescent plasma covid-19: immunopathology and its implications for therapy impact of immune enhancement on covid-19 polyclonal hyperimmune globulin therapy and vaccine development how immune complexes from certain igg nabs and any f(ab')(2) can mediate excessive complement activation immune complex-mediated tissue injury: a multistep paradigm antibody-dependent enhancement of sars coronavirus infection and its role in the pathogenesis of sars participants in the summit on dengue immune correlates of p. immune correlates of protection for dengue: state of the art and research agenda is covid-19 receiving ade from other coronaviruses? microbes infect dengue viruses and mononuclear phagocytes. i. infection enhancement by non-neutralizing antibody convalescent plasma: new evidence for an old therapeutic tool? clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study. lancet we would like to thank dr. arturo casadevall for critical review of the manuscript. key: cord-352985-5ccrkfsa authors: putter, jeffrey s.; seghatchian, jerard title: an update on covid-19 infection control measures, plasma-based therapeutics, corticosteroid pharmacotherapy and vaccine research date: 2020-09-04 journal: transfus apher sci doi: 10.1016/j.transci.2020.102934 sha: doc_id: 352985 cord_uid: 5ccrkfsa this communication provides a compilation on aspects of covid-19 infection control measures, describes the potential role of therapeutic plasma exchange to reduce fatality rates, addresses precautions concerning dexamethasone pharmacotherapy and updates the current status on the availability of vaccines. as part of passive immunotherapy, it focuses on various blood derivatives. these include coronavirus neutralising antibodies extracted from different sources to be administered as a pure hyper concentrate intramuscularly or for upgrading and standardising the specific potency of high affinity antibodies. these processes are intended to compose standardised pooled bioproducts of corona convalescent plasma/cryosupernatant that are pathogen inactivated for additional safety by well-established uv technologies. for the best practice of optimising plasma exchange, hyper concentrate nab should be added to the cryosupernatant, which contains some of the active principles of corona convalescent plasma. the cryosupernatant apart from the high molecular weight viscous part of cold insoluble proteins that are removed, is equivalent to ccp, but makes it safer for general application. such a bioproduct is often used routinely for substitution therapy of thrombotic thrombocytopenic purpura. alternative resources of large-scale specific coronavirus antibodies warrant further exploration such as cadaveric donations. the early uses of therapeutic plasma exchange and low molecular weight heparin, for any clinical trial in development is warranted, in order to interdict the intense inflammatory/kinin driven cascade. because coronavirus positive patients are highly prone to thrombosis, thromboprophylaxis is necessary, even some time after recovery guided by the laboratory data. exchange, hyper concentrate nab should be added to the cryosupernatant, which contains some of the active principles of corona convalescent plasma. the cryosupernatant apart from the high molecular weight viscous part of cold insoluble proteins that are removed, is equivalent to ccp, but makes it safer for general application. such a bioproduct is often used routinely for substitution therapy of thrombotic thrombocytopenic purpura. alternative we are well cognizant of a nefarious strain of the coronavirus sars cov2 that has been eclipsing the globe causing unprecedented health and economic chaos to the current generation. in a spirit of oneness to combat this marauding infection, we are challenged to develop and harness an arsenal of well-validated tools for the purposes of precision diagnoses, monitoring and treatment. vigilance in these key areas is critical to protect our national health and economies against a formidable adversary. currently, a collaboration of all international scientific and medical entities is summoned in an effort to develop effective preventative strategies to slow down the spread of this virus. equally important are optimising therapeutics to reduce the morbidity and mortality associated with this infection run amok. certain wellknown interests have sought to downplay the seriousness of the coronavirus problem in order to advance their own political agendas. one notable unsubstantiated allegation is that up to 99% of patient cases are harmless when in reality, the serious illness rate has fluctuated up to 14%. multiplicities of dangerous complications attendant to the virus, which cannot be ignored, include sepsis, refractory acute respiratory distress syndrome and multi-organ dysfunction and the severe vascular insults of thromboembolism and stroke. the propagation of the disease is of particular concern to a vulnerable patient population over the age of 65 years and among the icu patients, wherein mortality rates may exceed 33% for the elderly. contemporaneously, there are some significant gaps in leadership, often sending misinformation to the public that threatens the capacity to safely reopen. one poignant example is non-compliance in wearing face masks. there is very fine line between encouraging liberty for all juxtaposed to being responsible members of the society; working in concert to follow the medical guidelines and conscientiously foster public health safety. given that the coronavirus has dangerous capacity to cause consequential morbidity and mortality in spite of contemporary therapeutic modalities, we need to aggressively pursue new treatment strategies within our armamentarium, one being therapeutic plasma exchange. as the virus can cause excess inflammatory mediators such as cytokines and chemokines to circulate, in theory it would be advantageous to exchange patients with fresh frozen plasma or convalescent plasma containing a fixed dose of coronavirus neutralising antibody, nab, if available from a donor. the perpetuation of the coronavirus onslaught interestingly appears to be non-seasonal in nature and quite pernicious, an apparent continuous wave of infection worldwide. of special concern to public health officials is the potential devastating impact of other viruses such as influenza in the fall superimposed on the coronavirus plague. this is especially an issue for those that are susceptible, not having received an influenza vaccination. the incidence of coronavirus disease activity in the population appears to fluctuate from partially controlled at lock down to exponential growth of virus dissemination upon opening. the latter problem is thought to be consequent to a lapse in executing effective infection control practices by some noncompliers, with consistency, in the public. historically, "passive immunotherapy" has a positive track record as in the treatment of the ebola virus; these are smaller observational studies but they encourage therapeutic use for coronavirus too [3, 4] . in consideration of the very low risk of complications of plasma exchange therapy, it is a very attractive first treatment option for coronavirus. this is practicable if the usage is standardised, such as the use of (1.) "a minipool of pathogen reduced ccp with an elevated dose of neutralising antibodies from at least two ccp donations" or as a second viable preparation alternative, (2.) namely a homogenous standardised cryosupernatant with a fixed potency of neutralising antibody, as previously proposed by one of us [2] . in order to apply the use of passive immunotherapy consistently, coronaplasma bioproducts for transfusion exchange should be standardised with respect to the level, potency and avidity of antibodies such as would be advantageous in the processing of minipool convalescent plasma [2] . moreover, the antibody levels of ccp can be boosted by hyperimmune covid antibodies obtainable by using a promising on line affinity column separation methodology or otherwise, as described earlier [2] ; and such a pure hyperimmune coronavirus antibody can be delivered by intramuscular injections with fixed potency and could be prioritised to healthcare workers initially. alternatively, the above purer hyperimmune products can be used for upgrading the antibody content of the potential pool of ccp or its cryosupernatant, that will be essential as the carrier of such bioproduct in therapeutic plasma exchange (tpe). incorporating these hyperimmune products in plasma exchange is believed to have potential therapeutic value. to this end, it is currently indeterminate precisely which components of exchange plasmas might be integral to modulate the hyperinflammatory and hypercoagulable states triggered by the virus. exploring further, some large-scale alternative practicable sources of coronavirus neutralising antibodies are warranted for implementation. one possibility is consented cadaveric donations, either from the serum or a pulmonary lavage solution, but clearly in advance of decomposition. such an approach, if proven to be viable, could alleviate the time and cost-pressure and the need for higher throughput that make developing therapeutic neutralising antibodies at the right dose a real challenge to overcome, given the limited availability of corona convalescent plasma. regarding the importance of continual quality and safety improvement strategies related to harvesting corona nab, all bioproducts for reinfusion need to have pathogen reduction whether used as hyperimmune immunoglobulins or using targeted affinity column processing methods. in fact, this is a current requirement for all human derived bioproducts for which safety practices are already in place. affinity column processing is a favoured option, to mainly enrich the useful circulatory corona autoantibodies. in concert with the manufacturers, sterilized bioproducts in ampule format could be produced immediately to satisfy demand. it is important to highlight that circulating coronavirus antigen has been identified by rna assays in some adults who recovered from the infection. as a result of the possibility of a transmissible virus, the implementation of pathogen reduction methodology therefore must become an integral part of all reinfusion programs as highlighted before [1, 2] . we propose early targeted therapeutic plasma exchange in combination with inflammatory t-cells, monocytes, neutrophils and macrophages. there is thought to be a predisposition to cytokine and/or chemokine release in selected patients triggering a dangerous hyperimmune reaction and associated severe cytopathic effects such as a rapidly progressive pulmonary edema and acute hypercoagulability [11] . one observation is that patients having the poorest medical outcomes after contracting the infection have a tendency to a higher viral load, an rnaemia as assessed by pcr but not always a consistent finding. paradoxically, those that develop the highest titers of high affinity antibodies against the virion appear to have the poorest outcomes compared to patients with less severe disease. there is ongoing research into this inter-individual variability in response to coronavirus infection; to determine why these factors appear to be associated with an increased risk of developing a hyperimmune response and heightened allo/autoimmunity in some patients [2, 5] and impairing special t-cell and macrophage function essential for host defenses and recovery from the virus and/or nosocomial infection [7] . by comparison, it is instructive to reflect historically about the role of corticosteroids and the 2009 influensa a h1n1v outbreak. when studied in that pandemic, very early treatment by corticosteroids of ards occurred in a selected cohort of patients, the french registry that specifically excluded confounding comorbidities other than obesity. statistical analyses showed no medical benefit and yielded poorer outcomes, excess deaths in association with the steroids [8] . this leads us to view extremely cautiously the current recommendation for dexamethasone to treat oxygen dependent covid-19 patients. moreover, an endorsement of the use of dexamethasone absent precise guidelines to initiate treatment is of concern. ostensibly, patient's that would recover absent steroids may receive it anyway and now the ones we propose to protect are exposed to indeterminate risk. nevertheless, the fact is that vaccines take time to be fully implemented on a large scale globally. even if any of the trials indicate that such a vaccine is potentially working, a key question is whether there is a durable protective response to promote herd immunity and for how long. it is for this reason that it is more than likely a requirement to implement several different types of effective candidate vaccines and our newer proposal of passive immunity through safer pooled ccp with an upgraded nab protocol in parallel. the covid-19 pandemic has created unique challenges all over the world for infection control. in retrospect, given the easy respiratory transmission of the virus, we should have: (1.) marshalled earlier efforts by the manufacturers to ramp-up production of n95 masks to effectively filter out the virus; and (2.) promoted national educational campaigns on how to properly wear masks. as the virus shall likely linger with us for some years to come, it would clearly be productive to institute these two simple control measures going forward; current vaccine trials appear promising in the hope to abort the covid-19 pandemic but are not a panacea. the vaccine is likely to be ineffective for some and non-administered by many for various personal reasons. as a result, we need to encompass a range of therapeutic tools such as tpe, plasma derivatives and well-selected pharmacotherapies to supplant vaccines in order to treat the worst complications of this respiratory viral disease. a continuous wave of infection is already in place in some parts of europe and the usa. in the absence of a reliable vaccine, preparation of hyperimmune coronavirus nab must be pursued consistently and with standardisation from all sources. treatment for emerging viruses: convalescent plasma and covid-19 convalescent plasma, an apheresis research project by targeting and motivating the fully recovered covid-19 patients: a rousing message of clinical benefits to both donors/ recipients alike use of convalescent whole blood or plasma collected from patients recovered from ebola virus disease for transfusion as an empirical treatment during outbreaks ebola virus convalescent blood products: where we are now and where we may need to go covid-19, induced activation of haemostais, and immune reactions: can an auto-immune reaction contribute to the delayed severe complications observed in some patients low-cost dexamethasone reduces death by up to one third in hospitalised patients with severe respiratory complications of covid-1916. recovery trial 19#:~:text=the%20recovery%20trial%20involves%20many%20thousands%20of%20 doctors%2c dexamethasone for covid-19? not so fast early corticosteroids in severe influenza a/h1n1 pneumonia and acute respiratory distress syndrome safety and immunogenicity of the chad)x1ncov-19 vaccine against sars-cov-2: a preliminary report of a phase1/2, single-blind, randomized controlled trial. www.thelancet estimating the extent of asymptomatic covid-19 and its potential for community transmission: systematic review and meta-analysis pathophysiology, transmission, diagnosis, and treatment of coronavirus disease 2019 (covid-19) key: cord-305074-wz17u4e7 authors: fernandez, javier; gratacos-ginès, jordi; olivas, pol; costa, montserrat; nieto, susana; mateo, dolors; sánchez, maría belén; aguilar, ferran; bassegoda, octavi; ruiz, pablo; caballol, berta; pocurull, anna; llach, joan; mustieles, maría jesús; cid, joan; reverter, enric; toapanta, nestor david; hernández-tejero, maría; martínez, josé antonio; claria, joan; fernández, carlos; mensa, josé; arroyo, vicente; castro, pedro; lozano, miquel title: plasma exchange: an effective rescue therapy in critically ill patients with coronavirus disease 2019 infection date: 2020-08-24 journal: crit care med doi: 10.1097/ccm.0000000000004613 sha: doc_id: 305074 cord_uid: wz17u4e7 infection by severe acute respiratory syndrome coronavirus-2 can induce uncontrolled systemic inflammation and multiple organ failure. the aim of this study was to evaluate if plasma exchange, through the removal of circulating mediators, can be used as rescue therapy in these patients. design: single center case series. setting: local study. subjects: four critically ill adults with coronavirus disease 19 pneumonia that failed conventional interventions. interventions: plasma exchange. two to six sessions (1.2 plasma volumes). human albumin (5%) was used as the main replacement fluid. fresh frozen plasma and immunoglobulins were administered after each session to avoid coagulopathy and hypogammaglobulinemia. measurements and main results: serum markers of inflammation and macrophage activation. all patients showed a dramatic reduction in inflammatory markers, including the main cytokines, and improved severity scores after plasma exchange. all survived to icu admission. conclusions: plasma exchange mitigates cytokine storm, reverses organ failure, and could improve survival in critically ill patients with coronavirus disease 2019 infection. s ome patients affected by coronavirus disease 2019 (covid-19) develop severe inflammation and progressive organ failure that threaten survival. laboratory tests in these patients usually show elevation of inflammatory and coagulation markers (c-reactive protein [crp] , d-dimer) and data of macrophage activation (elevation of triglycerides, lactate dehydrogenase [ldh] , and ferritin) (1) (2) (3) (4) . corticosteroids and other immunosuppressive agents have been proposed in this setting (2) (3) (4) (5) (6) (7) . however, some patients do not respond to this therapy and develop multiple organ failure. therapeutic plasma exchange removes endogenous and exogenous inducers of the systemic inflammatory response (pathogen-associated molecular pattern and damage-associated molecular pattern) and proinflammatory mediators (cytokines and reactive oxygen species) that are involved in the pathogenesis of organ failure (8) . sporadic case reports suggest that therapeutic plasma exchange can be an effective rescue therapy in patients with hemophagocytic lymphohistiocytosis (9) or severe influenza a (h1n1) infection (10) . we report a case series of four critically ill patients infected by severe acute respiratory syndrome coronavirus 2 www.ccmjournal.org xxx 2020 • volume xx • number xxx (sars-cov-2) successfully treated with plasma exchange. therapeutic plasma exchange, two to six sessions, was performed with 5% albumin as the main replacement fluid (2/3). fresh frozen plasma (ffp) was used (1/3) at the end of the plasma exchange to avoid coagulopathy. iv immunoglobulin (ivig) was administered after each session (100 mg/kg) to prevent the hypogammaglobulinemia induced by the procedure. a 49-year-old man with obesity, hypertension, and type 2 diabetes was admitted to the hospital with fever and cough for 1 week and progressive shortness of breath within the previous 48 hours. oxygen saturation was 90% (fio 2 28%). laboratory tests indicated the following: serum crp 14.65 mg/dl, creatinine 1.44 mg/dl, ldh 449 u/l, triglycerides 752 mg/dl, ferritin 2,005 ng/ml, and d-dimer of 4,900 ng/ml. apart from lymphopenia (0.6 × 10^9/l), blood cell counts were normal, as well as procalcitonin levels and coagulation variables (supplementary table 1 , supplemental digital content 2, http://links.lww.com/ ccm/f804). arterial blood gases showed a pao 2 of 61.8 mm hg, and chest radiograph revealed bilateral lung infiltrates. polymerase chain reaction (pcr) for sars-cov-2 in nasopharyngeal smear was positive. lopinavir/ritonavir, hydroxychloroquine, interferon beta-1a, ceftriaxone, and linezolid were started. on day 2 of hospitalization, the patient was admitted to the icu due to progressive respiratory failure and required orotracheal intubation and mechanical ventilation. after intubation, norepinephrine was started. echocardiography disclosed mild ventricular dysfunction. troponin levels were normal. deep sedation and muscle relaxation were required in the first 48 hours after intubation due to severe hypoxemia (pafio 2 : 68) and poor adaptation to the ventilator. tocilizumab (400 mg) was administered on days 2 and 4 of icu admission. tracheotomy was performed 1 week after intubation. on day 9, he presented with high fever (41°c), sinus tachycardia with frequency-dependent left bundle branch block, and stage 2 acute kidney injury (aki; creatinine: 3.65 mg/dl). mild cardiac hypomotility persisted at echocardiography. inflammatory and macrophage activation parameters increased markedly ( fig. 1 table 2 , supplemental digital content 3, http://links.lww.com/ ccm/f805). cultures and body ct-scan ruled out bacterial or fungal infection or pulmonary thromboembolism. upon suspicion of hyperinflammatory state due to cytokine storm and macrophage activation like syndrome, therapeutic plasma exchange was started on day 10. four sessions were performed following an every other day schedule (days 10, 12, 14, and 16). the plasma volume exchanged by session was 4,900 ml. the patient condition improved in the following days: fever resolved 3 days following the fourth session of plasma exchange (day 19), renal, cardiac, and respiratory function normalized, and laboratory findings showed sustained improvement ( fig. 1 table 2 , supplemental digital content 3, http://links.lww.com/ccm/f805). plasma levels of most of the main cytokines decreased markedly after therapeutic plasma exchange (fig. 2) . severe myopathy and catheter-related infection by klebsiella pneumoniae 8 days after finishing plasma exchange were the most relevant problems during the rest of icu stay. on day 36, he was decannulated. the patient was discharged from the icu and from the hospital on days 38 and 51, respectively. a 59-year-old man was admitted to the hospital for a 10-day course of cough, fever, asthenia, and diarrhea. his medical history revealed a liver transplantation in 2007 treated with mycophenolate until current hospitalization, hypertension, insulin-dependent diabetes, and chronic kidney disease (creatinine 2.0 mg/dl). physical examination at admission was unremarkable except for lung basal crackles. oxygen saturation at that time was 85% at fio 2 21%. arterial blood gases demonstrated a pao 2 of 68.1 mm hg, and chest radiograph showed bilateral lung infiltrates. laboratory findings were as follows: crp 8.65 mg/dl, creatinine 2.01 mg/dl, ldh 429 u/l, triglycerides 344 mg/dl, ferritin 2,780 ng/ml, d-dimer 1,300 ng/ml. lymphopenia (0.4 × 10^9/l) was also present (supplementary table 1 , supplemental digital content 2, http://links.lww.com/ccm/f804). pcr for sars-cov-2 in nasopharyngeal smear was positive. he was started on lopinavir/ritonavir, hydroxychloroquine, azithromycin, ceftriaxone, and teicoplanin. twenty-four hours after admission, the patient developed progressive respiratory failure. in this setting, he was admitted to the icu and required invasive mechanical ventilation. after intubation, norepinephrine was started. an echocardiography disclosed moderate ventricular dysfunction suggestive of myocarditis. troponin levels were 6,963 ng/l. anakinra and stress dose hydrocortisone were started. although respiratory requirements improved over the next days, his overall condition worsened. he had persistent high fever without microbiological isolations and developed stage 3 aki in the setting of persistent cardiac dysfunction. serum markers of inflammation and macrophage activation remained high or worsened ( fig. 1 table 2 , supplemental digital content 3, http://links.lww.com/ccm/f805). tracheotomy was performed 9 days after icu admission. in this setting, therapeutic plasma exchange was started on day 10 of icu stay. only two sessions were performed due to moderate ffp transfusion reaction at the end of the second session (tachycardia, hypotension requiring an increase in norepinephrine doses, maculopapular rash, and increased leukocyte count). these two sessions were performed in consecutive days. the plasma volume exchanged by session was 5,000 ml. patient clinical condition improved 48 hours after the last session. fever resolved, cardiac and renal function normalized in few days, and regular laboratory tests ( fig. 1 table 2 , supplemental digital content 3, http://links.lww.com/ccm/f805) and plasma cytokines also improved (fig. 2) . the patient was decannulated 29 days after icu admission and was discharged from the icu and from the hospital on days 31 and 40, respectively. a 56-year-old man with obesity and hypertension was admitted to the hospital with bilateral lumbar pain for 4 days and diarrhea and loss of sensitivity and strength in the legs within the last 24 hours. at admission, he had fever (38°c) and was diaphoretic, tachycardic, tachypneic, and hypotensive. oxygen saturation at that time was 96% at fio 2 80%. laboratory test showed crp 22.76 mg/ dl, procalcitonin 2.93 ng/ml, creatinine 2.36 mg/dl, ldh 682 u/l, ferritin 1,396 ng/ml, troponin 224.7 ng/l, lactate 28.8 mg/dl, and d-dimer greater than 10.000 ng/ml (supplementary table 1 , supplemental digital content 2, http://links.lww. com/ccm/f804). blood cell counts disclosed leukocytosis and thrombocytopenia (25.93 and 52.0 × 10 9 /l, respectively). echocardiogram was normal. an angio-ct-scan showed bilateral infiltrates suggestive of covid-19 pneumonia, lobar and segmental acute right pulmonary thromboembolism, and multiple nonocclusive arterial thrombosis in distal aortic arch, splenic artery, aortoiliac bifurcation, iliac arteries, and right femoral artery. pcr for sars-cov-2 in nasopharyngeal swab was positive. the patient was admitted to the icu for respiratory (high flow nasal cannula) and vasopressor support (norepinephrine: 0.56 ug/ kg/min). he was started on lopinavir/ritonavir, hydroxychloroquine, azithromycin, and piperacillin-tazobactam. iv dexamethasone and heparin sodium infusion were also initiated. after initial improvement, the patient presented symptoms of acute limb ischemia on day 14 and required urgent bilateral transpopliteal embolectomy. screen for thrombophilia factors identified positive immunoglobulin g (igg) anticardiolipin serum antibodies. on day 15, catastrophic antiphospholipid syndrome was diagnosed and table 2 , supplemental digital content 3, http://links.lww. com/ccm/f805) and plasma cytokines levels (fig. 2 ) decreased markedly. the patient was discharged from hospital 33 days after admission on steroid and low molecular weight heparin therapy. no more thrombotic events were observed. a 64-year-old man with alcoholic liver cirrhosis, hypertension, and type 2 diabetes was admitted to the hospital with fever (38.5°c) for 2 days. pcr for sars-cov-2 in nasopharyngeal swab was positive. treatment with lopinavir/ritonavir, hydroxychloroquine, azithromycin, interferon beta-1a, ceftriaxone, and teicoplanin was started. four days after hospital admission, he presented progressive respiratory failure (oxygen saturation of 90% at fio 2 80%) and was admitted to the icu for invasive mechanical ventilation. chest radiograph showed bilateral lung infiltrates. at that time, laboratory test disclosed crp 2.34 mg/ dl, creatinine 2.41 mg/dl, ldh 426 u/l, ferritin 464 ng/ml, and d-dimer 900 ng/ml (supplementary table 1 , supplemental digital content 2, http://links.lww.com/ccm/f804). in this setting, methylprednisolone was started (60 mg/d for 5 d). nine days after icu admission, a tracheostomy was performed. however, 10 days later, the patient developed grade 2 hepatic encephalopathy and stage 2 aki. laboratory tests also showed an increase in inflammatory and coagulation markers (wbc, crp, ferritin, and d-dimer) and deterioration of liver function (serum bilirubin 5.4 mg/dl, international normalized ratio 1.35). abdominal doppler ultrasonography ruled out vascular complications. test for hepatotropic virus were negative except for herpes 6 (pcr in blood was weakly positive). with the orientation of an acute on chronic liver failure triggered by sars-cov-2, therapeutic plasma exchange was started 26 days after icu admission. the plasma volume exchanged by session was 3,800 ml. after completing three sessions performed on days 26, 27, and 29, liver (bilirubin 1.8 mg/dl, inr 1.18) and renal function improved, hepatic encephalopathy resolved, and inflammatory variables normalized (fig. 1) innate and adaptative responses that may lead to tissue damage and multiple organ failure (1, (3) (4) (5) 11) . high serum levels of proinflammatory cytokines (tumor necrosis factor [tnf]-alpha, interleukin [il]-1, il-2, il-6, il-17, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor) and chemokines (monocyte chemoattractant protein-1, macrophage inflammatory protein 1-alpha, il-8, interferon gamma-induced protein 10) have been reported in patients with severe acute respiratory syndrome (12) , middle east respiratory syndrome (mers) (13) , and also in severe covid-19 (1, 11) . macrophage activation syndrome may also occur (3). therapeutic plasma exchange removes inflammatory mediators from the systemic circulation and could ameliorate this covid-19-related cytokine storm and immunopathology, a hypothesis that has not been evaluated so far. our small case series supports this contention and suggests that plasma exchange combined to iv igg is an effective salvage therapy in patients with covid-19 pneumonia requiring critical care. three of our patients presented multiple organ failure despite having received conventional therapies that included antiviral and anti-inflammatory drugs. furthermore, two of them had analytical and clinical data of macrophage activation like syndrome. the initiation of therapeutic plasma exchange was temporarily associated with a marked clinical improvement with resolution of fever, improvement of renal and vascular function, decrease in sequential organ failure assessment and acute physiology and chronic health evaluation scores, and amelioration of inflammatory markers including variables of macrophage activation such as serum ferritin and triglyceride. a fourth patient was treated with therapeutic plasma exchange due to a catastrophic antiphospholipid syndrome induced by sars-cov-2 that was refractory to anticoagulation. importantly, systemic inflammatory markers decreased, and thrombotic events definitively resolved. our study also suggests that critically ill covid-19 patients show an hyperinflammatory state that can be mitigated by therapeutic plasma exchange. plasma levels of tnf-alpha and of other proinflammatory cytokines and chemokines were extremely high in samples taken before plasma exchange. this treatment effectively decreased levels of the great majority of cytokines and chemokines, therefore attenuating cytokine storm. it is important to remark that therapeutic plasma exchange was performed in our four patients using 5% albumin as the main replacement fluid. albumin is the main transporter and the main antioxidant and free-radical scavenger of human plasma (14) . ffp was administered at the end of each session to prevent coagulopathy. ivigs were also administered after each session to prevent the development of hypogammaglobulinemia. this apheresis approach was safe with just one patient developing a moderate transfusion reaction related with ffp infusion. in summary, our small case series suggests that therapeutic plasma exchange is an effective recue therapy in critically ill patients with covid-19 infection who do not respond to conventional therapies. this treatment was safe, ameliorated cytokine storm, reversed organ failure, and improved survival in very severe covid-19 patients. a randomized controlled trial, the recambio plasmatico (rep)-covid (clinicaltrials. gov identifier: nct04374539), is currently ongoing to confirm or reject our hypothesis. dr. sanchez disclosed work for hire. dr. reverter disclosed off-label product use of plasma exchange. dr. arroyo received funding from grifols. dr. lozano's institution received funding from terumo bct and received funding from grifols and cerus. the remaining authors have disclosed that they do not have any potential conflicts of interest. for information regarding this article, e-mail: jfdez@clinic.cat. clinical features of patients infected with 2019 novel coronavirus in wuhan, china risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus diwww.ccmjournal.org xxx 2020 • volume xx • number xxx sease 2019 pneumonia in wuhan, china the role of cytokines including interleukin-6 in covid-19 induced pneumonia and macrophage activation syndrome-like disease covid-19: immunopathology and its implications for therapy uk: covid-19: consider cytokine storm syndromes and immunosuppression chinese clinical trial registry: a multicenter, randomized controlled trial for the efficacy and safety of tocilizumab in the treatment of new coronavirus pneumonia (covid-19) interleukin-1 receptor blockade is associated with reduced mortality in sepsis patients with features of macrophage activation syndrome: reanalysis of a prior phase iii trial the mechanisms of action of plasma exchange role of plasma exchange, leukocytapheresis, and plasma diafiltration in management of refractory macrophage activation syndrome use of therapeutic plasma exchange as a rescue therapy in 2009 ph1n1 influenza a-an associated respiratory failure and hemodynamic shock dysregulation of immune response in patients with covid-19 in wuhan, china plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity human serum albumin, systemic inflammation, and cirrhosis key: cord-324908-ptlpsnfo authors: cao, huiling; shi, yuan title: convalescent plasma: possible therapy for novel coronavirus disease 2019 date: 2020-05-02 journal: transfusion doi: 10.1111/trf.15797 sha: doc_id: 324908 cord_uid: ptlpsnfo nan t he recent coronavirus disease 2019 (covid19) epidemic is spreading all over the world. by march 23, over 300,000 patients had been confirmed and almost 15,000 died because of covid-19. more than 200,000 cases have been confirmed in other countries and regions outside china. italy is the worst affected country in europe. so far, no specific effective treatment has been developed for covid-19 except for meticulous supportive care including critical care and organ support when necessary. convalescent plasma might be a potential treatment for covid-19. 1 convalescent plasma refers to a plasma therapy based on plasma or plasma derivatives obtained from donors who have survived previous infections by developing antibodies and infusing into newly infected individuals. 2 the precise action mechanism of convalescent plasma therapy is not fully stated. there are some assumptions: first and foremost, the assumption is that convalescent plasma contains protective antibodies by neutralizing the pathogen, eventually leading to its eradication from the blood circulation. rapid viral clearance would prevent further replication and the stimulus for the cytokine cascade. the level of anti-ebola virus immunoglobulin g (igg) titers was found to be associated with a delay in the peak of viral replication in a lethal ebola virus-infected mouse model. 3 another assumption is that convalescent plasma can convey other healing factors, such as preventing excess vascular leakage, procoagulant or antifibrinolytic factors, restoring the endothelium glycocalyx. 2, 4 convalescent plasma plays an important role as one of the treatments for many viral infections when vaccines or other specific treatments are not available. convalescent plasma has been applied more than 100 years (the first well documented was the spanish flu in 1917-1919). 2 there is an urgent need to have protective measures for nonexposed populations, prophylaxis for exposed but not yet infected populations, and experimental therapy for attacked individuals. [5] [6] [7] [8] [9] [10] [11] [12] each of those situations including the early spanish flu have proposed the use of convalescent plasma therapy. evidence shows that it is also effective in infectious diseases such as lassa fever, 13 argentine hemorrhagic fever, 14 measles, 15 and sin nombre virus. 16 public health of england and the international severe acute respiratory and emerging infection consortium 17 put forward that convalescent plasma could be a promising specific treatment for serious middle east respiratory syndrome (mers), and further evaluation is needed in human clinical trials. 18 the world health organization (who) announced in september 2014 that serum from people who are convalescing from infection with the ebola virus can be used to treat new patients. 19 twice tested negative for ebola virus rna by molecular techniques (the two samples for ebola virus rna testing should be taken at least 48 hours apart, and the test results should be negative on each sample) , and aged between 18 years old and 55 years old, could be considered as potential convalescent plasma donors. a dosage of 400 to 500 ml of convalescent plasma was given in two doses of 200 to 250 ml each, separated from two different whole blood donations. for pediatric convalescent plasma transfusion, a dose of 10 ml/kg could be used based on the considerations of blood volume. donors needed to be seronegative of hiv, hepatitis b virus, hepatitis c virus, syphilis, and other locally transmitted infections. 19 convalescent plasma has been used to treat several viral infections, including sars, avian influenza a (h5n1), influenza a (h1n1), mers, and ebola virus. although many studies have reported the efficacy and safety of convalescent plasma infusion in the treatment of various infections, due to the lack of large-scale, randomized, well-designed, and prospective clinical trials, we tend to consider convalescent plasma as an "empirical" therapy. 20 many studies have shown that convalescent plasma can effectively reduce viral load and increase antibodies to inhibit virus replication. nevertheless, subsequent trials about convalescent plasma showed different results. 13 the characteristics of primary study are described in table 1 . see table s1 (available as supporting information in the online version of this paper) for detailed data (https://data.mendeley. com/datasets/n8w9n7rgz3/1). a retrospective comparative study showed a shorter hospital stay after convalescent plasma therapy in sars patients who deteriorated despite ribavirin and high-dose steroid therapy (74% vs. 19%; p = 0.001). compared with five people who died in the continuing high-dose methylprednisolone group, there were no deaths in the plasma group (p = 0.049). 6 a case report by wong et al. 21 stated that a 57-year-old woman infected with sars improved gradually in clinical signs and symptoms after receiving a single 200-ml dose of convalescent plasma by 15 days after symptoms onset, suggesting that convalescent plasma combined with antiviral drugs and a corticosteroid may be an available option for the treatment of sars infection. 21 a study reported recovery of three patients infected with sars who developed severe progression and failed to respond to the ribavirin or methylprednisone. the study shows that viral load dropped from 495 × 10 3 , 76 × 10 3 , or 65 × 010 3 copies/ ml to zero or one copy/ml 1 day after transfusion, and anti-sars-cov immunoglobulin m (igm) and igg also increased in a time-dependent manner following convalescent plasma transfusion. 22 two cases of convalescent plasma were reported. both showed absolute reductions in viral load. one shows that the h7n9 virus was undetectable, the number of lymphocytes had been normalized by 4 days after infusing 200 ml of convalescent plasma with a titer of 80, and a computed tomography scan of the consolidation in the left lung had improved markedly after 12 days. 23 another case report provided by zhou et al., found that the virus load was reduced from 1.68 × 105 to 1.42 × 104 copies per milliliter during the first 8 hours and was undetectable within 32 hours after infusing convalescent plasma. 9 a look-back study suggests that patients with spanish influenza pneumonia who received transfusion with influenza-convalescent human blood products may have experienced a clinical reduction in the risk of death. the overall crude case fatality rate was 16% (54/336) among treated patients and 37% (452/1219) among controls. a significant absolute reduction in the case fatality rate was observed in the patients treated within 4 days (19%; 28/) compared with 4 days or later (59%; 49/83). therefore early definitive therapy is of great significance for pneumonia and hypoxia. 24 in 2012, rockman et al. 8 conducted an animal experiment in which ferrets were exposed to lethal doses of highly pathogenic influenza h5n1, infused with hyperimmune serum at three different times (24 hr before or 24 hr after virus exposure at the onset of fever [>40°c] or immediately before the earliest expected onset of significant clinical signs [estimated from previous studies as day 3 after exposure]). a total of 12 ferrets were included in the treatment group. the purpose of this study was to investigate the effects of convalescent plasma infusion at different time periods on survival. all four animals transfused hyperimmune serum 24 hours before virus exposure survived and were generally well, with slight weight loss. we can conclude that the greatest benefit was derived from passive immunization provided immediately before contact with an infectious dose of virus, compared to buffer controls or h3n2 nonhomologous hyperimmune serum. a prospective cohort study designed by hung et al. 25 in which patients received a single 500-ml dose of convalescent plasma with an antibody titer greater than 160. mortality in the treatment group was significantly lower than in the nontreatment group ( controlled trial reported that patients who received immune plasma and standard care for severe influenza showed a nonsignificant reduction in the mortality rate. 26 in 1995, a small study reported that eight patients received 150 to 400 ml of convalescent plasma and seven survived, for a case fatality rate of 12.5% in comparison to 80% in patients without convalescent plasma treatment. 29 however, van griensven et al. 4 performed a nonrandomized comparison study in which patients received two consecutive transfusions of 200 ml to 250 ml with unknown levels of antibody titer. eighty-four patients received convalescent plasma was not associated with a significant improvement in survival. from day 3 to day 16 after diagnosis, the risk of death was 31% (26/84 patients) in the convalescent plasma group and 38% (158/418 patients) in the control group (risk difference, −7 percentage points; 95% ci, −18 to 4). in conclusion, the transfusion of up to 500 ml of convalescent plasma with unknown levels of neutralizing antibodies in 84 patients with confirmed ebola virus infection was not associated with a significant improvement in survival. further clinical trial is worth performing. covid-19 belongs to the same coronavirus family as sars and mers. many life-threatening complications, such as acute respiratory distress syndrome, can occur during the viral mass replication phase. none of them had specific and effective treatment. according to previous studies and who recommendations, convalescent plasma might be used when a specific treatment is not available. according to the press conference of the joint prevention and control mechanism of the state council on february 28 (guangming net of china), up to 544 doses of plasma from convalescent covid-19 patients have been collected across the country and applied to 245 covid-19 patients. of the 157 covid-19 patients who received convalescent plasma therapy and were closely monitored for more than 48 hours, 91 cases showed improvement in clinical indicators and symptoms. the plasma therapy has proved to be safe and effective. neutralizing antibodies against the novel coronavirus have been identified in the plasma of convalescent patients. a pilot study that has just been reported shows that the convalescent plasma (titer ≥ 640) therapy was safe and could improve clinical symptoms and laboratory parameters. 30 we look forward to the release of relevant data about convalescent plasma applied in covid-19. more prospective comparative studies are needed to confirm the efficacy of convalescent plasma. no serious adverse events were associated with convalescent plasma treatment. 4, 5, 31 the most commonly reported mild adverse event was a brief "chill" reaction with a transient hyperpyrexia after the convalescent plasma transfusion. few patients develop transfusion-related adverse events such as phlebitis, generalized jaundice, or anaphylaxis. 24 a case report raised an association between transfusion-related acute lung injury and convalescent plasma. 32 in most viral illnesses, viremia peaks in the first week of infection. the patient then develops a primary immune response by day 10 to day 14, followed by virus clearance. arabi et al. 18 studied the time course of specific antibody response and found that those antibodies peaked 1 week after the inoculation and then began declining. therefore, convalescent plasma should be more effective when given early during the course of infections. sars patients whose clinical condition deteriorated after receiving ribavirin and methylprednisolone had a higher discharge rate by day 22, a shorter hospital stay, and a lower mortality rate when convalescent plasma was administered before day14 of illness onset. 5 in a study of patients with lassa fever in nigeria, all eight patients who received convalescent plasma before day 10 of illness recovered and survived, while only three of eight patients who received plasma after day 10 survived. 13 the first 3 weeks of symptom onset, there was an increase in the titer of igg and igm antibodies to sars-cov-2. all those patients achieved a seroconversion of igg or igm within 20 days after symptom onset. the median day of seroconversion for both igg and igm was 13 days (after symptoms onset). the igg levels in all the patients reached the platform in 6 days after the first positive points. it means that we should use convalescent plasma within 3 weeks after symptoms onset. 33 these findings suggest that early initiation of convalescent plasma treatment may be of critical importance to reduce mortality in patients with sars or other pathogen infection. 31 however, a report showed that, when convalescent plasma transfusions were initiated on the day of diagnosis or up to 2 days later, the risk of death on day 3 to day 16 was nonsignificant. 4 therefore, whether earlier use of convalescent plasma is better need more evidences. there are still some issues to consider in determining the advisability of implementing a large-scale convalescent plasma transfusion program: what is the optimal timing for infusion? how long after clinical resolution of symptoms is there a chance to obtain neutralization antibodies if any? what is the most effective frequency of administration of convalescent plasma? is clinical therapeutic effect related to antibody titer? is it more likely to benefit young children and pregnant women? how do we make efficient use of convalescent plasma? more physiological studies are needed to explore why convalescent plasma is superior to fresh plasma and convalescent whole blood. clinical research needs to explore the relationship between time (both extraction time and infusion time), volume, frequency, antibody titer and other healing factors, and clinical symptoms and signs of the patient. there is limited availability of eligible potential donors with sufficient levels of antibody. more large-scale, randomized, well-designed prospective studies are needed. of note, eight studies, including a randomized controlled trial, have been registered in the chinese clinical trial registry (http://www.chictr.org.cn), preparing for clinical research (chictr2000030690, chictr2000030627, chictr2000030557, chictr2000030381, chictr2000030312, chictr2000030046, chictr2000030039, chictr2000029850). convalescent plasma is now used as an empirical treatment in the absence of specific treatment for covid-19 and other dangerous viral infections, although its efficacy remains controversial. there are still some questions need to be explored. we look forward to more well-designed prospective studies. hc drafted the initial manuscript and reviewed the manuscript. ys critically reviewed the manuscript for important intellectual content. this work is attributed to children's hospital of chongqing medical university. convalescent plasma as a potential therapy for covid-19 use of convalescent plasma in ebola virus infection passive transfer of antibodies protects immunocompetent and immunodeficient mice against lethal ebola virus infection without complete inhibition of viral replication evaluation of convalescent plasma for ebola virus disease in guinea use of convalescent plasma therapy in sars patients in hong kong retrospective comparison of convalescent plasma with continuing high-dose methylprednisolone treatment in sars patients a study in scarlet-convalescent plasma for severe influenza the use of hyperimmune serum for severe influenza infections treatment with convalescent plasma for influenza a (h5n1) infection characteristics and outcome of mechanically ventilated patients with 2009 h1n1 influenza in bosnia and herzegovina and serbia: impact of newly established multidisciplinary intensive care units severe h1n1-associated acute respiratory failure in immunocompromised children anti-ebola virus antibody levels in convalescent plasma and viral load after plasma infusion in patients with ebola virus disease the use of lassa fever convalescent plasma in nigeria convalescent whole blood, plasma and serum in the prophylaxis of measles ribavirin, human convalescent plasma and anti-beta3 integrin antibody inhibit infection by sin nombre virus in the deer mouse model treatment of mers-cov: information for clinicians feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia use of convalescent whole blood or plasma collected from patients recovered from ebola virus disease for transfusion, as an empirical treatment during outbreaks. interim guidance for national health authorities and blood transfusion services. geneva: world health organization convalescent plasma: new evidence for an old therapeutic tool? treatment of severe acute respiratory syndrome with convalescent plasma successful treatment of avian-origin influenza a (h7n9) infection using convalescent plasma experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a taiwan hospital meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection immune plasma for the treatment of severe influenza: an open-label, multicentre, phase 2 randomised study anti-influenza immune plasma for the treatment of patients with severe influenza a: a randomised, double-blind, phase 3 trial the efficacy of convalescent plasma for the treatment of severe influenza. medrxiv treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients. international scientific and technical committee the feasibility of convalescent plasma therapy in severe covid-19 patients: a pilot study. medrxiv, preprint the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory metaanalysis acute respiratory distress syndrome after convalescent plasma use: treatment of a patient with ebola virus disease antibody responses to sars-cov-2 in covid-19 patients: the perspective application of serological tests in clinical practice the authors have disclosed no conflicts of interest. key: cord-274150-ukdha3ap authors: choi, jun yong title: convalescent plasma therapy for coronavirus disease 2019 date: 2020-09-03 journal: infect chemother doi: 10.3947/ic.2020.52.3.307 sha: doc_id: 274150 cord_uid: ukdha3ap convalescent plasma has been used for decades to prevent and treat a wide range of infectious diseases for which no specific treatment is available. the use of convalescent plasma involves transfusing plasma collected from patients who have recovered from a viral illness, in an attempt to transfer virus-neutralizing antibodies and confer passive immunity. in addition to the antiviral mechanisms of neutralizing antibodies, the immunomodulatory effects of plasma components could have benefits. several small and large-scale studies have shown the effects of convalescent plasma for the treatment of severe coronavirus disease 2019 (covid-19). in addition to transfusion-related side effects, unexpected side effects such as antibody-dependent enhancement (ade) may occur during convalescent plasma therapy, but early safety studies have not found any cases of ade among more than 5,000 participants. with historical precedents and recent clinical studies, convalescent plasma therapy should be considered as a candidate therapy for covid-19 given the limited effectiveness of antiviral drugs and lack of a vaccine. a system to secure safe collection and use of convalescent plasma should be developed as a response to the pandemic. further clinical trials should be conducted to determine the safety and efficacy of convalescent plasma therapy concurrently with its clinical use. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection has become a global concern, and the world health organization (who) declared coronavirus disease 2019 (covid19) a public health emergency of international concern on january 30, 2020, and a pandemic on march 11, 2020 [1] . between december 31, 2019 and july 29, 2020, more than 16 million cases of covid-19 were reported worldwide, including more than 660,000 deaths. most patients with covid-19 have mild disease and recover without any treatment. however, older individuals with comorbidities such as diabetes mellitus, hypertension, and respiratory or cardiovascular disease are at greater risk of complications and mortality [2, 3] . to overcome the covid-19 pandemic, the development of effective therapeutic agents and vaccines is crucial. however, as of august 3, 2020 , there are few therapeutic agents that have been approved for the treatment of covid-19, and there are no vaccines [4] . remdesivir is the only antiviral agent that showed efficacy in a randomized controlled trial [5] , and dexamethasone is another medication that showed efficacy in severe covid-19 patients [6] . however, having these medications is not enough to overcome the covid-19 pandemic. given the lack of therapeutic options for covid-19 and vaccines, historical interventions for emerging infectious diseases have remerged as options for disease control. given its rapid acquisition, convalescent plasma therapy has been considered as an emergency intervention in several pandemics, including the spanish flu, severe acute respiratory syndrome coronavirus (sars-cov-1), and west nile virus, and more recently, ebola virus [7] [8] [9] . during the current covid-19 pandemic, multiple studies of varying sizes have been conducted on convalescent plasma therapy for covid-19. the purpose of this review is to provide the theoretical rationale, summarize the evidence of safety and effectiveness, and discuss considerations for clinical application of convalescent plasma therapy. in the early twentieth century, convalescent sera were used to stem epidemics of viral diseases such as poliomyelitis, measles, mumps, and influenza [10] . a retrospective metaanalysis of eight studies on the use of convalescent sera involving 1703 patients during the 1918 h1n1 influenza virus pandemic suggested that those who received serum had lower mortality [11] . during the 2009 -2010 h1n1 influenza pandemic, convalescent plasma was used to treat patients with severe h1n1 infection requiring critical care [12] . a study with 93 participants showed that treatment of severe h1n1 2009 infection with convalescent plasma reduced respiratory tract viral load, serum cytokine response, and mortality. during the 2013 ebola epidemic in west africa, a nonrandomized study showed significantly longer survival for those treated with convalescent whole blood compared to those who received standard treatment [13] . however, another nonrandomized study on ebola virus disease did not show the survival benefit of convalescent plasma therapy [14] . convalescent plasma therapy has also been used for treating coronavirus diseases such as severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers). a study from a hospital in hong kong evaluated the efficacy of convalescent plasma therapy in the treatment of patients with sars in 2003 [15] . a higher day-22 discharge rate was observed among patients who were given convalescent plasma before day 14 of illness and among those who were pcr positive and seronegative for coronavirus at the time of plasma infusion. during the mers epidemic in south korea, convalescent plasma therapy was performed for several mers patients, and a study suggested that donor plasma with a neutralization activity of a plaque reduction neutralization test (prnt) titer ≥1:80 should be used for convalescent plasma therapy [16] . although large-scale randomized controlled trials have not yet been performed, and most studies did not evaluate neutralizing activities of used convalescent plasma, previous experiences on convalescent plasma therapy for the treatment of emerging infectious diseases provide us with important historical precedents that this intervention might be useful for confronting the covid-19 epidemics. convalescent plasma therapy is a passive antibody therapy that involves the administration of antibodies against a given pathogen to a susceptible individual for the purpose of preventing or treating an infectious disease. neutralizing antibodies have been considered essential in the effects of convalescent plasma therapy, and the efficacy of this therapy was associated with the titer of neutralizing antibodies in the convalescent plasma [17, 18] . in addition to the antiviral mechanisms of neutralizing antibodies, immunomodulatory effects of plasma components could have additional benefits [18] . during apheresis, in addition to neutralizing antibodies, other proteins such as anti-inflammatory cytokines, clotting factors, natural antibodies, defensins, pentraxins, and other undefined proteins are obtained from donors [19] . in this sense, transfusion of convalescent plasma in infected patients may provide further benefits such as immunomodulation via amelioration of severe inflammatory response. several uncontrolled case series of convalescent plasma therapy for the treatment of patients with covid-19 have suggested a possible benefit [20] [21] [22] [23] . multiple studies have evaluated the effects of convalescent plasma therapy for patients with covid-19 (table 1 ) [20, [23] [24] [25] [26] [27] . the reports showed that convalescent plasma therapy induced reduction in viral loads and improvement of laboratory markers and clinical signs. most studies were non-randomized, non-controlled studies, and the participants had severe or life-threatening disease. given encouraging historical precedents and the lack of proven effective therapy for covid-19, clinical studies on the use of convalescent plasma as a treatment option for covid-19 are ongoing in many countries. as of august 3 2020, a total of 132 studies evaluating the role of convalescent plasma in covid-19 are registered on clinicaltrials.gov. the first randomized trial of convalescent plasma therapy for covid-19 patients was conducted in china [28] . to evaluate the efficacy and adverse effects of convalescent plasma therapy for patients with covid19 , an open-label, multicenter, randomized clinical trial was performed in seven medical centers in wuhan, china. the study included 103 participants with severe or life-threatening covid-19. to ensure the therapeutic potency of the convalescent plasma, only the plasma units with a spike protein receptor-binding domain (s-rbd)-specific immunoglobulin g (igg) titer of at least 1:640 were used in the study. however, because the covid-19 epidemic in china was contained while enrolment was in progress, the trial was terminated before it reached its targeted sample size of 200 patients. hence, it was underpowered and many comparisons between the convalescent plasma group and the control group were not statistically significant. although a recent systematic review showed uncertainty regarding the effectiveness of convalescent plasma for individuals with covid-19 [29] , ongoing studies including randomized controlled trials will provide further evidence of the effectiveness and safety of convalescent plasma therapy for covid-19. there are several concerns about the use of convalescent plasma therapy for covid-19 patients. transfusion of plasma may cause transfusion-related adverse events, including minor adverse events such as fever, nausea, allergic reactions, transmission of blood-borne pathogens, and some severe adverse events such as transfusion-related acute lung injury (trali), transfusion-associated circulatory overload (taco), and antibody-dependent enhancement (ade) [10, 30] . taco and trali are the leading causes of transfusion-related fatalities, and specific therapies are unavailable [31] . taco and trali are syndromes of acute respiratory distress that occur within 6 hours of blood transfusion. the pathophysiology of both syndromes is not fully understood. in taco, cardiac or renal impairment and positive fluid balance may precede circulatory overload. trali often presents as bilateral pulmonary edema with little evidence of circulatory overload. trali may be caused by preceding inflammation, antileukocyte antibodies, and biological response modifiers. because covid-19 itself can induce lung injury, the underlying lung injury associated with covid-19 further complicates the differential diagnosis of taco and trali, and may increase the risk of taco and trali in critically ill patients. ade may occur when antibodies to the virus fail to efficiently neutralize the virus. previous studies showed that the binding of virions to non-neutralizing or sub-neutralizing antibodies could lead to more efficient viral uptake into the target cell in fcγ receptor or through complement-mediated mechanisms, leading to enhanced viral replication [32, 33] . ade has been reported for various viral diseases including dengue, zika, influenza, and respiratory syncytial virus infection. the ade phenomenon has been demonstrated for sars-cov-1 using in vitro and animal models [34] . however, to date, there has been no evidence of ade in reported cases and clinical trials on coronaviruses [7, [20] [21] [22] [23] 28] . a united states (us) study of 5,000 hospitalized adults with severe or life-threatening covid-19, with 66% in the intensive care unit, provided safety data on convalescent plasma therapy [30] . the incidence of all serious adverse events (saes) in the first four hours after transfusion was <1%, including a 0.3% mortality rate. of the 36 reported saes, 25 were assessed as possibly or definitely transfusion related, including mortality (4 cases), taco (7 cases), trali (11 cases), and severe allergic transfusion reactions (3 cases). however, only 2 (of 36) saes were judged as definitely related to the convalescent plasma transfusion by the treating physician. the study suggested that convalescent plasma therapy for hospitalized patients with covid-19 was relatively safe. the us fda also provide suggested donor eligibility criteria which stipulate that donors should satisfy the following conditions: (1) evidence of covid-19 documented by a laboratory test either by a diagnostic test (e.g., nasopharyngeal swab) at the time of illness or a positive serological test for sars-cov-2 antibodies after recovery, if prior diagnostic testing was not performed at the time that covid-19 was suspected. (2) either one of the following (i) complete resolution of symptoms at least 28 days prior to donation; or (ii) complete resolution of symptoms at least 14 days prior to donation, and negative results for covid-19 either from one or more nasopharyngeal swab specimens or by a molecular diagnostic test from blood. (3) male donors, or female donors who have not been pregnant, or female donors who have been tested since their most recent pregnancy and results were interpreted as negative for human leukocyte antigen (hla) antibodies. (4) sars-cov-2 neutralizing antibody titers, if available (when measurement of neutralizing antibody titers is available, the us fda recommends neutralizing antibody titers of at least 1:160. a titer of 1:80 may be considered acceptable if an alternative matched unit is not available. when measurement of neutralizing antibody titers is not available, it is suggested that a retention sample from the convalescent plasma donation be stored so that the antibody titers can be determined at a later date. the korea centers for disease control (kcdc) has also developed donor eligibility criteria as detailed below [36] : (1) an individual who has recovered from covid-19 can donate plasma at least 14 days after being cured of covid-19. negative results of real-time reverse transcription polymerase chain reaction for sars-cov-2 using a respiratory specimen should be confirmed if the plasma collection is performed less than 28 days after the donor has been cured of covid-19. infectious disease specialists and laboratory medicine specialists should confirm that the donor is completely cured of covid-19 at the point of plasma collection. (2) before plasma collection, the donor's body weight, medical history, social history, physical examination, and laboratory tests must be evaluated to determine whether the individual is suitable to serve as a plasma donor. the minimum standard for age (17 to 69 years old, donors over 65 years of age who have experience of blood donation from 60 to 64 years old), body weight (50 kg or more for men, 45 kg or more for women), and hemoglobin level 12 g/dl or more) should be met. some patients who recover from viral diseases may not have high titers of neutralizing antibodies, which are crucial for the effectiveness of convalescent plasma therapy [13, 14] . a study showed that virus-specific igg levels in asymptomatic covid-19 patients were significantly lower than those in symptomatic patients in the acute phase [37] . of asymptomatic individuals, 93.3% and 81.1% had reductions in igg and neutralizing antibody levels, respectively, during the early convalescent phase, as compared to 96.8% and 62.2% of symptomatic patients, respectively. forty percent of asymptomatic individuals, and 12.9% of symptomatic individuals, became seronegative for igg in the early convalescent phase. another study showed that sars-cov-2-specific neutralizing antibody titers were low for the first 7 -10 days after symptom onset and increased after 2 -3 weeks [38] . the median peak time for neutralizing antibodies was 33 days after symptom onset. the titers in 93·3% of the patients declined gradually over the 3-month study period. another study evaluated serological reactivity in plasma from 436 convalescent plasma donors with a history of disease compatible with covid-19 in england [39] . the study showed that antibody levels declined over 3 months following the diagnosis. because historical experience of the use of convalescent plasma therapy suggests that the neutralizing activity may correlate with the efficacy of convalescent plasma, the measurement of sars-cov-2 neutralizing antibody titers should be considered, if available. however, laboratory methods for evaluating neutralizing antibody titers, such as the prnt, microneutralization assay, and pseudovirus neutralization assay, are not routinely performed in hospital settings. however, as several reports suggest that sars-cov-2 specific igg titers correlate with neutralizing antibody titers [38, 40] , the sars-cov-2specific igg titer should be measured in the convalescent plasma. to perform convalescent plasma therapy for covid-19, development of a system with an adequate infrastructure is required. a population of donors who have recovered from the disease and can donate convalescent serum need to be recruited. blood banking facilities to process apheresis are necessary. essential assays, including serological assays to detect sars-cov-2 antibodies in serum, and virological assays to measure viral neutralization, should be performed. to perform the assays for neutralization antibodies, the necessary laboratory support to perform these assays should be implemented. protocols for safe collection and use of convalescent plasma should be developed. clinical trials to assess the efficacy, safety, and immunologic responses should be combined with clinical use. regulatory review and approval should be performed in a timely manner. pharmaceutical companies should try to develop highly purified preparations containing a high titer of neutralizing antibodies against sars-cov-2, which is preferable to convalescent plasma. the preparation may be safer and have higher neutralization activity; however, the development of the hyper-immunoglobulin preparation takes many months. with historical precedents and recent clinical studies, convalescent plasma therapy should be considered as a candidate intervention for covid-19, given the limited evidence of effectiveness of antiviral agents and the lack of a vaccine. a system to secure safe collection and use of convalescent plasma should be developed as a response to the pandemic. further scientific studies on the safety and effectiveness of convalescent plasma therapy should be explored through clinical trials that can be established concurrently with the clinical use of convalescent plasma therapy. coronavirus disease (covid-19) pandemic ns; china medical treatment expert group for covid-19. clinical characteristics of coronavirus disease 2019 in china age-related morbidity and mortality among patients with covid-19 interim guidelines on antiviral therapy for covid-19 dexamethasone in hospitalized patients with covid-19 -preliminary report convalescent plasma study group. the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis ebola virus disease: an emerging and re-emerging viral threat west nile virus neutralization by us plasma-derived immunoglobulin products the convalescent sera option for containing covid-19 meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection evaluation of convalescent whole blood for treating ebola virus disease in evaluation of convalescent plasma for ebola virus disease in guinea use of convalescent plasma therapy in sars patients in hong kong challenges of convalescent plasma infusion therapy in middle east respiratory coronavirus infection: a single centre experience convalescent plasma transfusion for the treatment of covid-19: systematic review convalescent plasma in covid-19: possible mechanisms of action plasma therapy against infectious pathogens, as of yesterday, today and tomorrow effectiveness of convalescent plasma therapy in severe covid-19 patients treatment of 5 critically ill patients with covid-19 with convalescent plasma treatment with convalescent plasma for critically ill patients with severe acute respiratory syndrome coronavirus 2 infection use of convalescent plasma therapy in two covid-19 patients with acute respiratory distress syndrome in korea effect of convalescent plasma therapy on viral shedding and survival in patients with coronavirus disease 2019 treatment of coronavirus disease 2019 (covid-19) patients with convalescent plasma covid-19 plasma task force. mortality reduction in 46 severe covid-19 patients treated with hyperimmune plasma. a proof of concept single arm multicenter trial clinical efficacy of convalescent plasma for treatment of covid-19 infections: results of a multicenter clinical study effect of convalescent plasma therapy on time to clinical improvement in patients with severe and life-threatening covid-19: a randomized clinical trial convalescent plasma or hyperimmune immunoglobulin for people with covid-19: a living systematic review early safety indicators of covid-19 convalescent plasma in 5,000 patients transfusion-associated circulatory overload and transfusion-related acute lung injury viral-induced enhanced disease illness molecular mechanism for antibody-dependent enhancement of coronavirus entry immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates recommendations for investigational covid-19 convalescent plasma guidelines for collecting convalescent plasma of covid-19 clinical and immunological assessment of asymptomatic sars-cov-2 infections longitudinal dynamics of the neutralizing antibody response to sars-cov-2 infection zambon m; nhs blood and transplant convalescent plasma testing group. convalescent plasma treatment for sars-cov-2 infection: analysis of the first 436 donors in england humoral and circulating follicular helper t cell responses in recovered patients with covid-19 key: cord-328352-l1q4uvxl authors: borlongan, mia c.; borlongan, maximillian c.; sanberg, paul r. title: the disillusioned comfort with covid-19 and the potential of convalescent plasma and cell therapy date: 2020-08-25 journal: cell transplant doi: 10.1177/0963689720940719 sha: doc_id: 328352 cord_uid: l1q4uvxl coronavirus disease 2019 or covid-19 is highly infectious, which can lead to acute and chronic debilitating symptoms, as well as mortality. the advent of safe and effective vaccines or antiviral drugs remains distant in the future. practical public health measures, such as social distancing, hand washing, and wearing a face mask, are the current recommended guidelines by the centers for disease control and prevention for limiting the spread of the virus. weakened immune system and aberrant inflammation represent a major pathological symptom of covid-19 patients. based on the unique immunomodulatory properties of both convalescent plasma and stem cells, we discuss here their potential use for treating covid-19. may be likely accounted for by human behavior, especially those who do not take seriously the centers for disease control and prevention (cdc)-recommended public health practices of social distancing and proper hygiene, i.e., washing of hands with soap and water. moreover, the virusassociated microdroplets can stay airborne for up to 3 h 6 , necessitating the need for wearing a face mask to protect ourselves and others. the combination of 6-feet social distancing, 20-s hand washing, and wearing n-95 or other cdc-recommended face masks has been reported to curve the spread of virus 7, 8 , but quantifying the risk of reducing these strategies remains debatable. notwithstanding, the cdc continues to stress practicing social distancing, hand washing, and wearing a face mask in order to inhibit future deaths from the virus. otherwise, we are living in our "disillusioned comfort," similar to the misguided security blanket we afford to negative consequences of smoking, gambling, and riding a motorcycle. a historical review of virus-related pandemics reminds us of the 1918 spanish flu, which infected 500 million and estimated to have killed about 17 million to 50 million people 9 . the spanish flu resembles covid-19: both are respiratory viruses with the precursor viruses mutating in animals thereafter infecting humans, and with fast-progressive cases from infection to symptom onset with viral consolidation in the lung and eventually mortality primarily from pneumonia 9-12 . eerily enough, the key promoters of such high mortality rate of spanish flu were overcrowding and poor hygiene-the same two factors that define our disillusioned comfort for covid-19. human behavior to the spanish flu outbreak stands as the major determinant for the pattern of the pandemic's high mortality rate 13 . again, the same human response, or lack thereof, that faces covid-19. a spanish flu vaccine was not available during the 1918 pandemic, with the virus remaining largely unknown for almost a century when the molecular underpinnings of the virus were finally characterized in 2005 14 . similar to covid-19, the 1918 spanish flu was first detected in the month of january, but after the second wave in august of that year, the number of cases dropped significantly and almost disappeared by the year's end. without a vaccine, the rapid decline in infection and mortality after the second of the two lethal waves of spanish flu was attributed to better treatment of pneumonia and the virus mutating to a less dangerous strain. three key lessons from the spanish flu pandemic are worthy of consideration. first, those who survived the first wave developed immunity against the virus; thus, these individuals were immune to the second wave of the spanish flu, providing us a glimpse on the future of passive immunitybased treatment against the virus. indeed, clinical trials have recently been initiated using plasma collected from recovered covid-19 patients based on the concept that these individuals have likely harbored strong immune responses such as antibodies that target the proteins found on covid-19 15 . second, that the end of spanish flu cases by 1920 might have signaled the eradication of the pandemic may not be completely true. evidence of slow-progressive cases of spanish flu has been linked to secondary bacterial pneumonia, possibly affecting the brain, which results in neurological disorders, in particular encephalitis lethargica, that recorded its outbreak in the 1920s, in children 16 , which was suggested to manifest in adulthood as parkinson's disease 17 but such a pathological link being merely coincidental has been debated 18 . similarly, the potential long-term consequences of covid-19 to brain disorders, and other comorbid diseases, warrant critical research avenues as we tackle this pandemic and its associated not-so-apparent mini outbreaks. third, that overcrowding in military camps and lack of sanitation due to poverty during world war i contributed to spanish flu pandemic draws our attention to the key pressing issue of human behavior. regenerative medicine may play a key role in treating covid-19. the ongoing clinical trials of infusing convalescent plasma stand as a potent cell therapy for covid-19 patients. this investigational treatment uses plasma that contains antibodies to severe acute respiratory syndrome coronavirus 2 or sars-cov-2, the virus that causes covid-19. plasma therapy entails the use of plasma or specific, fractioned, antibodies, along with other immunoglobulins (igs) and possibly other therapeutic molecules harvested from immunized individuals or convalescent persons 19 . plasma therapy has a long history of safety, and even efficacy, since the spanish flu in 1917-1918 19, 20 . while the mechanism of action of plasma therapy remains not fully understood, the functional benefits have been largely ascribed to the purified neutralizing antibodies of the convalescent plasma 19, 20 . a meta-analysis of english-language journals from 1918 to 1925 reveals that the overall mortality rate was 16% among treated patients compared to 37% among controls, indicating difference in fatality between the treatment and control groups at 8% to 26% 20 . note that the meta-analysis study 20 was based on only eight studies with many methodologic limitations, including lack of blinding, randomization, or placebo-controlled arm. subsequent to the spanish flu pandemic, the treatment with convalescent plasma has been studied in similar outbreaks of respiratory infections, such as the 2003 sars-cov-1 epidemic, the 2009-2010 h1n1 influenza virus pandemic, and the 2012 mers-cov epidemic 19, 20 . whereas small clinical trials and anecdotal accounts of convalescent plasma suggest safety and efficacy of plasma treatment in these previous outbreaks 21 , its potency in covid-19 requires carefully designed and rigorous assessment of these clinical trials as discussed subsequently. the still limited evidence but massively accumulating interest in the safety and efficacy of convalescent plasma treatments may provide the basis for compassionate use of these therapies for covid-19 in the united states. indeed, a review of the literature from 2020 alone shows that there are already 152 publications, although most are review and commentary articles, and protocol descriptions, on the theme of convalescent plasma treatments for covid-19. notable clinical research reports include: (1) the small study on six chinese patients diagnosed with covid-19, who subsequently received abo-compatible convalescent plasma, demonstrating immediate increase of antibody titers in two patients and elimination of the virus in two other patients 22 . (2) another small study on ten severe covid-19 chinese patients revealed that nine patients displayed increased neutralizing antibody titers, and the viral load in seven patients becoming undetectable by day seven post-infusion of convalescent plasma, but only 3 exhibited improved respiratory function 23 . (3) another study on five critically ill covid-19 patients revealed viral loads decreased and became negative by day 12, and three patients were discharged by about 50 days, while 2 patients remained in stable condition at 37 days after convalescent plasma infusion 24 . (4) in six end-stage covid-19 patients, convalescent plasma effectively induced viral shedding with the sars-cov-2 rna undetectable by day 3 post-infusion of convalescent plasma, but five patients eventually died 25 ; (5) in south korea, two severe covid-19 patients treated with convalescent plasma with both showing favorable outcomes including being weaned from mechanical ventilators and/or extubated with sars-cov-2 negative after days 20-26 of convalescent plasma infusion 26 . from these five small clinical case reports, the following protocol specifics may require optimization in order to further assess the safety and efficacy of convalescent plasma treatment for covid-19. (1) the small number of patients is clearly a limiting factor in all these studies. (2) once the number of patients to be enrolled is increased, there is a need for proper control and randomization of treatments, as well as blinding of the treating physicians. (3) the timing of convalescent plasma infusion and its doses and the need for booster shots will require careful considerations. for example, in duan's study 23 , patients were infused with convalescent plasma at around 16 days of symptom onset, while in shen's study 24 , patients received convalescent plasma at around 21 days after symptom onset. the recognition of positive outcomes may depend on the early initiation of convalescent plasma infusion and may be the need for subsequent booster treatment. additionally, in duan's study 23 , one dose of 200 ml of convalescent plasma with the neutralizing antibody titers above 1:640 was specified. in shen's study 24 , it was 400 ml of convalescent plasma with the neutralizing antibody titer set at 1:1000. finding the optimal timing and dose will improve the safety and efficacy of convalescent plasma. (4) related to the timing of convalescent plasma infusion, the severity of the disease is likely to impact on the treatment outcomes. in all these five clinical studies, it is understandable that because convalescent plasma treatment remains experimental, the target population is initially the severe or critically ill patients, who have high mortality and worst morbidity, which may mask the potential functional benefits of the treatment. future treatments may need to enroll those mild covid-19 patients or those who are in the early stages of the disease. (5) the treatment profile of the patients will need to be carefully charted, noting the drugs and ventilator supportive care among others, in order to delineate the true effects of convalescent plasma infusion or the possibility of additive effects of convalescent plasma with antiviral agents and immunomodulatory drugs. (6) the patient's preexisting health condition is equally an important factor in treatment outcomes and disease progression. in addition to covid-19, immune-related diseases may worsen the presenting pathology, which will likely require not only eliminating sars-cov-2 but also addressing the multifactorial manifestations of disease. (7) the age, gender, ethnicity, genetic factors, and other pertinent patient profiles will need to be factored into the final analysis of the treatment outcomes as this will further guide optimization of the convalescent plasma treatment. clearly, there remain several factors in order to advance the widespread use of convalescent plasma for treating covid-19. however, at the minimum, the transparency in reporting the detailed methods and treatment outcomes will ensure rigorous assessment of the true functional effects of convalescent plasma. a glance at the clinicaltrials.gov website reveals 173 ongoing clinical trials on the use of convalescent plasma for covid-19. recognition of the factors mentioned earlier will allow direct comparisons of protocols and safety and efficacy readouts across these clinical trials and further evaluate the potential of convalescent plasma for covid-19. as noted earlier, plasma therapy involves the use of convalescent plasma, but also the plasma-active components specifically igs. under the guise of passive immunity against pathogens in viral epidemics, convalescent plasma is considered the first-line passive immunotherapy since the 1918 spanish flu and the modern viral epidemics (2003 sars-cov-1, 2009-2010 h1n1, and 2012 mers-cov), and now covid-19 27, 28 . the last several decades have witnessed advancements in molecular technology toward the purification and manufacture of plasma-derived ig as a drug therapy 27, 28 . toward this end, human ig formulations have been advanced as the second line of passive immunotherapies against these diseases 27, 28 . the rationale of human ig for intravenous use (ivig) is thus based on the similar etiology and inflammatory pathogenesis of sars-cov-2 infection to diseases for which the use of ivig has already been approved by the fda 29 . according to clinicaltrials.gov, there are 40 trials using ig for covid-19. similar to convalescent plasma treatment, the mechanism of action ascribed to ig therapy is to boost the immune response of the body against sars-cov-2. along this line of boosting the immune system of covid-19 patients, mesenchymal stem cells (mscs) possess secretory functions of robust immunomodulatory biologics [30] [31] [32] [33] [34] . because covid-19 manifests a massive inflammatory pathology in the lung, characterized by pulmonary edema and an over-reactive immune response, which can lead to hypoxia, respiratory distress, and lung damage, finding a treatment designed to sequester this aberrant inflammation and immune response may prove beneficial against covid-19 33 . accordingly, cognizant that abrogating covid-19 may depend on the patients' properly functioning immune system, the transplantation of mscs has been proposed to propel the host immune system to effectively eliminate the virus. mscs display potent antiinflammatory and immunomodulatory properties. indeed, our group and many others have shown that mscs exert anti-inflammatory effects and even repair lung damage 34 . although mscs have been the major cell type for cell therapy in covid-19, non-msc stem cells, such as umbilical cord blood monocytes 35, 36 , that similarly target the inflammatory and immune pathways and promote other regenerative mechanisms (e.g., tissue repair), are also being tested. interestingly, intravenous administration of mscs has the natural preponderance to migrate preferentially to the lungs allowing for the first-pass treatment of pulmonary diseases 33, 34 , such as covid-19. moreover, the long track record of safety profile of mscs for hematologic diseases may expedite their clinical entry for novel indications; indeed, clinicaltrials.gov reveals 31 ongoing studies using stem cell therapy for covid-19. preliminary reports in china and the united states suggest solid safety outcomes in patients who have received mscs and their exosomes 31, 32 . together with convalescent plasma and ig treatments, stem cell therapies may offer an innovative approach in fostering immunomodulatory regulation of covid-19. moreover, although the lung stands as the main pathological organ in covid-19, recent studies have implicated extra-lung organs, including the heart and the brain, as well as the gut, suggesting that targeting these organs may prove beneficial in treating the disease. indeed, combining the envisioned vaccines and immunomodulatory treatments with novel strategies, such as altering the gut microbiome 37-41 , will likely enhance the overall prognosis of covid-19. science and medicine may take time to advance a vaccine, antiviral drug, convalescent plasma treatment, and ig and stem cell therapies to the clinic. mortality rate ranges from 0.1% to as high as 10%, with the elderly (>65 years of age) and those with preexisting immune-related and inflammatory diseases (hypertension, diabetes, heart diseases), showing higher incidence of mortality [42] [43] [44] . given the morbidity not only in the acute stage, but the realization of equally devastating long-term effects, including cardiovascular and cerebrovascular consequences [45] [46] [47] [48] [49] , warrants immediate action to at least lessen the spread of the virus. without access to the vaccine and other proven therapies, just like in 1918 spanish flu era, the best course of action for the meantime is practicing social distancing and good hygiene, which represents a logical strategy to curve the course and to address the disillusioned comfort of covid-19. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the author(s) received no financial support for the research, authorship, and/or publication of this article. mia c. borlongan https://orcid.org/0000-0003-3223-8374 lung cancer mortality associated with smoking and smoking cessation among people living with hiv in the united states. version 2 gender-related differences in the characteristics of problem gamblers using a gambling 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cord blood-derived monocytes improve cognitive deficits and reduce amyloid-b pathology in psapp mice monocytes are essential for the neuroprotective effect of human cord blood cells following middle cerebral artery occlusion in rat a gut feeling about stroke reveals gut-brain axis' active role in homeostasis and dysbiosis gut microbiome: lactation, childbirth, lung dysbiosis, animal modeling, stem cell treatment, and cns disorders a gutsy move for cell-based regenerative medicine in parkinson's disease: targeting the gut microbiome to sequester inflammation and neurotoxicity gutting the brain of inflammation: a key role of gut microbiome in human umbilical cord blood plasma therapy in parkinson's disease model probiotics and prebiotics as a therapeutic strategy to improve memory in a model of middle-aged rats characteristics and outcomes of coronavirus disease patients under nonsurge conditions time-to-death approach in revealing chronicity and severity of covid-19 across the world a geroscience perspective on covid-19 mortality neurologic complications of covid-19 covid-19 and the cerebro-cardiovascular systems: what do we know so far? potential effects of coronaviruses on the cardiovascular system: a review excess neurological death in new york city after the emergence of covid-19 a systematic review of neurological symptoms and complications of covid-19 key: cord-322714-s0wge7o4 authors: joyner, michael j.; bruno, katelyn a.; klassen, stephen a.; kunze, katie l.; johnson, patrick w.; lesser, elizabeth r.; wiggins, chad c.; senefeld, jonathon w.; klompas, allan m.; hodge, david o.; shepherd, john r.a.; rea, robert f.; whelan, emily r.; clayburn, andrew j.; spiegel, matthew r.; baker, sarah e.; larson, kathryn f.; ripoll, juan g.; andersen, kylie j.; buras, matthew r.; vogt, matthew n.p.; herasevich, vitaly; dennis, joshua j.; regimbal, riley j.; bauer, philippe r.; blair, janis e.; van buskirk, camille m.; winters, jeffrey l.; stubbs, james r.; van helmond, noud; butterfield, brian p.; sexton, matthew a.; diaz soto, juan c.; paneth, nigel s.; verdun, nicole c.; marks, peter; casadevall, arturo; fairweather, delisa; carter, rickey e.; wright, r. scott title: safety update: covid-19 convalescent plasma in 20,000 hospitalized patients date: 2020-07-19 journal: mayo clin proc doi: 10.1016/j.mayocp.2020.06.028 sha: doc_id: 322714 cord_uid: s0wge7o4 abstract objective to provide an update on key safety metrics after transfusion of convalescent plasma in hospitalized covid-19 patients, having previously demonstrated safety in 5,000 hospitalized patients. patients and methods from april 3 to june 2, 2020, the us fda expanded access program for covid-19 convalescent plasma transfused a convenience sample of 20,000 hospitalized patients with covid-19 convalescent plasma. results the incidence of all serious adverse events was low; these included transfusion reactions (n=89; <1%), thromboembolic or thrombotic events (n=87; <1%), and cardiac events (n=680, ∼3%). notably, the vast majority of the thromboembolic or thrombotic events (n=55) and cardiac events (n=562) were judged to be unrelated to the plasma transfusion per se. the seven-day mortality rate was 8.6% (8.2%, 9.0%), and was higher among more critically-ill patients relative to less ill counterparts, including patients admitted to the intensive care unit vs. not admitted (10.5% vs. 6.0%), mechanically ventilated vs. not ventilated (12.1% vs. 6.2%), and with septic shock or multiple organ dysfunction/failure vs. those without dysfunction/failure (14.0% vs. 7.6%). conclusion these updated data provide robust evidence that transfusion of convalescent plasma is safe in hospitalized patients with covid-19, and support the notion that earlier administration of plasma within the clinical course of covid-19 is more likely to reduce mortality. coronavirus disease 2019 continues to be a world-wide pandemic, and the number of deaths attributed to covid-19 in the us at the time of this writing (~110,000) exceed that of any other nation in the world 1 . the overall case fatality rate for diagnosed covid-19 ranges from about 4% to greater than 50% 2-6 , with higher mortality rates observed in more critically ill patients. in response to the covid-19 outbreak in the us and reportedly high case-fatality rates, the us food and drug administration (fda) in collaboration with the mayo clinic and national blood banking community developed a national expanded access program (eap) to collect and distribute covid-19 convalescent plasma. historical precedent indicates that human convalescent plasma is a viable option for mitigation and treatment of covid-19 7, 8 . the premise of human convalescent plasma therapy is that plasma of recently-infected and currently-recovered covid-19 patients contains anti-viral antibodies and other bioactive elements that can be used to treat patients with covid-19. convalescent plasma has a strong historical record of some efficacy during acute infectious pandemics 7, 9 . as recently summarized 7 , convalescent plasma represents a promising treatment strategy with strong historical precedence, biological plausibility, and limited barriers for rapid development and deployment of this investigational therapy. recently, our investigation of key safety indicators in 5,000 patients transfused with covid-19 convalescent plasma demonstrated an incidence of transfusion-related serious adverse events (sae) of less than 1% and a mortality rate of 14.9% 10 . these early indicators suggest that transfusion of convalescent plasma is safe in hospitalized adults with covid-19. because an additional 15,000 hospitalized patients have been transfused with convalescent plasma under the purview of the eap, a subsequent safety update is warranted. these new data may provide novel insights into the incidence of emerging adverse events associated with covid-19, including thromboembolic 11, 12 and cardiac events 13 . further, these data may provide better understanding of the clinical features contributing to the seven-day mortality rate among transfused patients with covid-19. thus, we analyzed key safety metrics following transfusion of convalescent plasma in 20,000 hospitalized adults with severe or life-threatening covid-19. these data represent a deeper and larger analysis relative to our initial report of 5,000 transfused patients under the eap. we hypothesized that both the seven-day mortality rate and the number of serious adverse events related to the transfusion of convalescent plasma would continue to be low. additionally, we hypothesized that higher mortality rates would be observed in more critically-ill patients. as described previously 10 , the program is a fda-initiated, national, multicenter, open-label eap in hospitalized adults that had (or were judged to have high risk of progression to) severe or life-threatening covid-19. the us covid-19 convalescent plasma eap was conducted as a pragmatic treatment study, empowering local acute care facilities to use the emerging best evidence for care while allowing for administration of convalescent plasma. it was conducted within a modified clinical trial framework: all participants were allowed access to convalescent plasma per the discretion of the treating physician/ principal investigator given the nature of the pandemic and the lack of any effective therapies at the time of design. it was the intent a priori to create a control comparator group to determine potential efficacy using patients hospitalized with covid-19 infections during the same time period. this decision was made after collaboration with the us fda. this report is only on safety of the cp for the initial 20,000 subjects. a future publication will discuss potential efficacy. between the date of initial institutional review board (irb) approval of the eap (april 1 st 2020) and june 11 2020, more than 20,000 patients were transfused with covid-19 convalescent plasma, figure 1 . the mayo clinic irb served as the central irb and empaneled an independent data and safety monitoring board (dsmb) to oversee the safety analyses. written informed consent was obtained from the participant or a legally-authorized representative prior to enrollment, or with use of emergency consent procedures recommended by the us fda and approved by the mayo clinic irb. the protocol was modified to allow inclusion of incarcerated participants once it became clear that this group was especially vulnerable and at risk. eligible patients were aged 18 years or older, hospitalized with a laboratory confirmed diagnosis of infection with severe acute respiratory syndrome coronavirus 2 (sars-cov-2), and had (or were judged by a healthcare provider to be at high risk of progression to) severe or life-threatening covid-19. the clinical symptoms defining severe or life-threatening covid-19 are outlined in table 1 . as described previously 10 , abo-compatible covid-19 convalescent plasma had no minimum neutralizing-antibody titer level and was donated by recently-recovered, covid-19 survivors. approximately 200 -500 ml of convalescent plasma was administered intravenously according to institutional transfusion guidelines. web-based, standardized data reporting surveys were completed to assess clinical status of patients at regular time intervals (four-hours and seven-days after convalescent plasma transfusion) using the research electronic data capture system (redcap, v.9.1.15 vanderbilt university, nashville, tn) 14, 15 . serious adverse event reporting. separate redcap data collection forms were used to report each sae that occurred within seven days following the convalescent plasma transfusion. two primary data capture forms were used to report saes: a transfusion form and a sae form. serious adverse events which occurred during the time window beginning at the onset of the plasma transfusion and including the four-hour time-period following the transfusion were reported on the transfusion form. by definition, the primary saes related to transfusion (including transfusion associated circulatory overload [taco] and transfusion-related acute lung injury [trali]) occurred within six hours of the transfusion. all transfusion-related saes occurred within four hours of the transfusion and thus, were all reported on the transfusion form. all other saes were reported on the sae form. the attribution scale used by treating physicians for evaluating sae relatedness to convalescent plasma transfusion included unrelated, possibility related, probably related, or definitely related. all transfusion-related saes were independently adjudicated over the course of the study by the ind sponsor (mjj) and trained designee (amk) using the national healthcare safety network biovigilance component hemovigilance module surveillance protocol as a conceptual framework 16 . statistics. data presented in this safety report may undergo additional data quality control measures as the study continues. the cumulative incidence of each of a series of saes was summarized using a point estimate and 95% score confidence interval (ci), as outlined in table 2 . to assess mortality, time (in days) between transfusion and mortality was examined using the kaplan meier product limit estimator. participants were censored at their last known vital status and all reported mortalities through seven days were used to estimate the survival function. data were censored at 0.25 days for patients who did not have follow-up beyond the initial report at four hours post transfusion at time of the analysis. for patients who expired within 24 hours, a survival time of 0.5 days was assigned. precise time of day for key events was not recorded in the data collection system; thus, these imprecise time estimates were used. the point estimate and 95% ci for mortality were estimated at day seven based on the estimated survival function. all analyses and graphics were produced with r version 3.6.2 (vienna, austria). from april 3 to june 11, 2020, a total of 30,117 patients were enrolled in the eap and a total of 21,987 enrolled patients received a covid-19 convalescent plasma transfusion (figure 1) . data from the first 5,000 transfused patients have been reported previously 10 . this update reports data from 20,000 patients including the initial 5,000 and subsequent 15,000 transfused patients. by june 2, 2020, a total of 20,000 patients had been transfused with covid-19 convalescent plasma, thus, 7-day mortality data is presented for all 20,000 patients. table 1 . the patient enrollment indicates a wide age range of hospitalized patients with covid-19, consistent with prior cdc published data 17 . the study population was diverse, with 20% of patients being african american, nearly 35% hispanic and 5% asian. the recruitment of a diverse population has improved over the time course of the study. nearly 40% of our subjects were women. most of the patients enrolled were overweight or obese, consistent with early reports of risk 18 . nearly all of the patients had severe or life-threatening covid-19, by design of the investigational protocol. nearly two-thirds had respiratory failure as well as dyspnea as a primary symptom. most were hypoxic and nearly half had pulmonary infiltrates. at least one-third had severe respiratory compromise. equal numbers appeared to have multi-organ failure or septic shock, but these were a small percentage of the total population. table 2 . our report is not a comprehensive summary of all risks associated with hospitalization of covid-19 but did assume that convalescent plasma potentially could cause life-threatening cardiac events and thrombotic events, so these were collected with an underlying assumption of attribution. within four hours of completion of the covid-19 convalescent plasma transfusion, 146 saes classified as transfusion reactions were reported (<1% of all transfusions). of these saes, there were 83 non-mortality events reported, with 37 reports of transfusion-associated circulatory overload (taco), 20 reports of transfusionrelated acute lung injury (trali), and 26 reports of severe allergic transfusion reaction. of the saes reported within four hours of plasma transfusion, there were 63 mortalities (0.3% of all transfusions) and 13 of these mortalities were judged as related (possibly, n=12; probably, n=1; definitely, n=0) to the transfusion of covid-19 convalescent plasma. within seven days of completion of the covid-19 convalescent plasma transfusion, 1,136 other saes were reported. of these saes, 87 thromboembolic or thrombotic events were reported, 406 sustained hypotensive events requiring intravenous pressor support were reported, and 643 patients suffered a cardiac event. notably, the vast majority of the thromboembolic or thrombotic complications (n=55) and cardiac events (n=569) were judged to be unrelated to the plasma transfusion. table 2 . over the first seven days after the covid-19 convalescent plasma transfusion, a total of 1,711 deaths were observed. the overall seven-day mortality rate was 8.56% (95% ci: 8.18%, 8.95%), figure 2 . the seven-day mortality rate was higher among the sickest of our critically-ill patients, including patients admitted to the intensive care unit (icu) vs. not admitted to the icu (10.5% vs. 6.0%), mechanically ventilated vs. not mechanically ventilated (12.1% vs. 6.2%), and those with septic shock or multiple organ dysfunction/failure vs. without septic shock or multiple organ dysfunction/failure (14.0% vs. 7.6%). in this safety update of the us convalescent plasma expanded access program of 20,000 hospitalized patients in the us with severe or life-threatening covid-19, the overall frequency of saes classified as attributable or likely secondary to convalescent plasma transfusion continued to be low (<1% of all transfusions) and the seven-day mortality rate in this extremely high risk cohort was 8.6%. despite the potential risks associated with plasma transfusion in critically-ill patients 16, 19 , these data provide continued optimism for the safety of covid-19 convalescent plasma. although thrombotic and thromboembolic events are emerging clinical complications of covid-19 11, 12, 20 , our data demonstrate a low rate (<1%) of these events within the first seven days after covid-19 convalescent plasma transfusion. cardiac events represent another novel clinical concern of covid-19 13 , particularly in the context of open-label use of experimental treatments such as hydroxychloroquine 21 . in aggregate, adverse cardiac events occurred in ~3% of patients transfused with covid-19 convalescent plasma. the vast majority of adverse cardiac events of interest were deemed unrelated to the plasma transfusion (88%) by the treating physicians. collectively, these data suggest that transfusion of covid-19 convalescent plasma per se does not demonstrably increase the risk of adverse cardiac events. we note that the incidence of trali and taco reported in this study of 0.18% and 0.10%, respectively is significantly lower than those reported in previous studies, which ranged from 5-8% and 1-4%, respectively 22 , despite the fact that many of the covid-19 patients receiving plasma were critically ill and likely at risk for clinical conditions which mimic taco. limitations. although this study was not designed to evaluate efficacy of convalescent plasma, we note with optimism that after 20,000 transfused patients the seven-day mortality rate (8.6%) is lower than the mortality rate observed in the first 5,000 transfused patients (12.0%), table 2 . while the mortality rate has fallen (figure 2) , we note that the clinical characteristics of the transfused patients in the eap have shifted toward less critically-ill patients and lower proportions of apparent "rescue therapy". no therapy has been introduced into clinical use during this time period which reduces (to our knowledge) mortality in hospitalized patients with covid-19. we postulate that several potential explanations may explain the observed decline in mortality. the ability and success of the us healthcare community at managing hospitalized covid-19 patients is likely improving. second, the blood banking communities have enhanced the availability of convalescent plasma such that more patients received plasma earlier in their hospital course compared to the initial cohort of 5000 participants. the more expeditious delivery of plasma to patients reflects improved logistics for the collection, deployment and use of convalescent plasma, and a larger covid-19 recovered population that may be potential plasma donors. in this regard it is remarkable that there was no system in place for convalescent plasma use in march 2020 and yet within months the nation is now able to meet most of the demand, despite complex logistics 23 . given the historical experience that antibody therapies are most effective when given earlier and that convalescent plasma has reduced mortality in prior epidemics 7 , the lower mortality in more recently treated patients would be consistent with greater efficacy from earlier use. finally, as recovered covid-19 patients were more quickly recruited for convalescent plasma donation, the plasma may contain higher levels of neutralizing antibodies or other bioactive elements. however, additional data are required to evaluate these potential explanations for the observed trends. data from the first 20,000 patients transfused with covid-19 convalescent plasma demonstrate that use of convalescent plasma is safe and carries no excess risk of complications. indeed, convalescent plasma may be associated with improvement in survival, however, this report does not establish efficacy. additionally, our data demonstrate that the us health care system is improving in its care for those hospitalized for covid-19 including managing those critically-ill patients with multiple comorbidities included in these analyses. overall, the mortality observed has fallen with our observations and continued use of convalescent plasma. given the accelerating deployment of this therapy, these emerging data provide early safety indicators of convalescent plasma for covid-19 treatment and suggest research should shift focus from safety toward determining the efficacy of convalescent plasma. for disease control and prevention. coronavirus disease 2019 (covid-19): cases in us the many estimates of the covid-19 case fatality rate 15-day mortality and associated risk factors for hospitalized patients with covid-19 in wuhan, china: an ambispective observational cohort study clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the covid-19 in critically ill patients in the seattle region -case series the convalescent sera option for containing covid-19 a serological survey on neutralizing antibody titer of sars convalescent sera meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? early safety indicators of covid-19 convalescent plasma fibrinolysis shutdown correlates to thromboembolic events in severe covid-19 infection autopsy findings and venous thromboembolism in patients with covid-19 cardiac involvement in a patient with coronavirus disease 2019 (covid-19) research electronic data capture (redcap)--a metadata-driven methodology and workflow process for providing translational research informatics support the redcap consortium: building an international community of software platform partners centers for disease control and prevention. the national healthcare safety network (nhsn) manual: biovigilance component v2.5 hospitalization rates and characteristics of patients hospitalized with laboratoryconfirmed coronavirus disease 2019-covid-net, 14 states clinical characteristics of covid-19 in new york city covid-19 convalescent plasma: now is the time for better science confirmation of the high cumulative incidence of thrombotic complications in critically ill icu patients with covid-19: an updated analysis urgent guidance for navigating and circumventing the qtc-prolonging and torsadogenic potential of possible pharmacotherapies for coronavirus disease 19 (covid-19) taco and trali: biology, risk factors, and prevention strategies deployment of convalescent plasma for the prevention and treatment of covid-19 6%) 80 years or older 650 (10.5%) 1,858 (13.9%) 60 (14.1%) 2,568 (12.8%) gender women 2,254 (36.3%) 5,360 (40.1%) 163 (38.2%) 7,777 (38.9%) men 3,935 (63.3%) 7,953 (59.5%) 264 (61.8%) 12,152 (60.8%) 17.6%) 2,356 (17.8%) 62 (14.6%) 3,246 (17.7%) overweight 1,413 (30.1%) 3,695 (28.0%) 120 (28.2%) 5,228 (28.5%) obese 2%) lung infiltrates > 50% the ratio of partial pressure of arterial oxygen to fraction of inspired oxygen ratio 56% (8.18%, 8.95%) clinical status no icu admission 17% (5.75%, 6.61%) mechanical ventilation (n = 6,337) 767 12.10% (11.32%, 12.93%) clinical symptoms no mof or septic shock (n = 17,081) 62% (7.23%, 8.03%) mof or septic shock abbreviations: icu, intensive care unit; mof, multiple organ failure or dysfunction. a point estimate of related serious adverse event incidence relative to 20,000 transfusions. b sustained hypotension included events requiring intravenous pressor support. c cardiac events included ventricular or atrial fibrillation or arrhythmia requiring treatment, and cardiac arrest. d these data were previously published 10 and reported for comparison with current data. key: cord-305130-vz72ldbo authors: keil, shawn d.; ragan, izabela; yonemura, susan; hartson, lindsay; dart, nicole k.; bowen, richard title: inactivation of severe acute respiratory syndrome coronavirus 2 in plasma and platelet products using a riboflavin and ultraviolet light‐based photochemical treatment date: 2020-05-14 journal: vox sang doi: 10.1111/vox.12937 sha: doc_id: 305130 cord_uid: vz72ldbo background and objective: severe acute respiratory distress syndrome coronavirus‐2 (sars‐cov‐2), the causative agent of coronavirus disease 2019 (covid‐19), is a member of the coronavirus family. coronavirus infections in humans are typically associated with respiratory illnesses; however, viral rna has been isolated in serum from infected patients. coronaviruses have been identified as a potential low‐risk threat to blood safety. the mirasol pathogen reduction technology (prt) system utilizes riboflavin and ultraviolet (uv) light to render blood‐borne pathogens noninfectious, while maintaining blood product quality. here, we report on the efficacy of riboflavin and uv light against the pandemic virus sars‐cov‐2 when tested in both plasma and platelets units. materials and methods: stock sars‐cov‐2 was grown in vero cells and inoculated into either plasma or platelet units. those units were then treated with riboflavin and uv light. the infectious titres of sars‐cov‐2 were determined by plaque assay using vero cells. a total of five (n = 5) plasma and three (n = 3) platelet products were evaluated in this study. results: in both experiments, the measured titre of sars‐cov‐2 was below the limit of detection following treatment with riboflavin and uv light. the mean log reductions in the viral titres were ≥3·40 and ≥4·53 for the plasma units and platelet units, respectively. conclusion: riboflavin and uv light effectively reduced the titre of sars‐cov‐2 in both plasma and platelet products to below the limit of detection in tissue culture. the data suggest that the process would be effective in reducing the theoretical risk of transfusion transmitted sars‐cov‐2. in early december 2019, an unusual cluster of pneumonia cases caused by an unknown agent was observed in wuhan, china [1, 2] . the unknown agent, now known as severe acute respiratory syndrome coronavirus-2 (sars-cov-2) [3] , is the causative agent of coronavirus disease 2019 (covid-19). initially declared a public health emergency on 30 january 2020, the world health organization eventually reclassified covid-19 as a pandemic on 11 march 2020, citing the alarming rate at which the disease was spreading. the number of infected individuals that will contract this agent will almost certainly continue to rise until significant herd immunity has developed. sars-cov-2 is the seventh known human coronavirus, which also includes middle east respiratory syndrome coronavirus (mers-cov) and severe acute respiratory syndrome coronavirus (sars-cov), having mortality rates of 35á5% and 10%, respectively [4] . the other four species of coronaviruses have mainly been associated with causing cold-like symptoms [5] . the sudden appearance of sars-cov-2 in humans is believed to have been the result of a zoonotic transmission event, although the proximate intermediate host remains unknown [6, 7] . covid-19 has a range of symptoms that includes fever, fatigue, dry cough, aches, and laboured breathing to acute respiratory distress and possibly death. it has also been reported that many infected individuals remain asymptomatic [8, 9] , which has complicated public health efforts to contain the spread of the virus. unknown are the effects this virus may have on national blood supplies; however, the 2003 outbreak of sars-cov was shown to have a negative impact [10] . to date, there have been no known transfusion transmitted cases of sars-cov or mers-cov; however, the aabb considered mers-cov as an agent of concern [11] . during the 2003 outbreak of sars-cov, viral rna was detected in the serum of symptomatic patients [12] [13] [14] , and in another study, symptomatic mers-cov-infected patients showed viral loads up to 5-6 log rna copies/ml in their serum [15] . the author of mers-cov study also indicated that they were not able to recover infectious virus from those patient samples using cell culture, suggesting that transfusion transmission is a low risk [15] . similar to the previous coronavirus outbreaks, viral rna has been recovered from symptomatic sars-cov-2 patients [16, 17] . in late january 2020, the wuhan blood center began using real-time pcr to screen all blood donations and retrospectively found viral rna in 4 asymptomatic donors [18] . however, the us fda is not recommending the use of laboratory screening tests of asymptomatic donors given that respiratory viruses, including coronaviruses, are not known to be transmitted by blood transfusion. to date, there have been no reported cases of transfusion transmission of sars-cov-2 [19] . the mirasol pathogen reduction technology (prt) system was created to provide an additional layer of safety to blood products by reducing the risk of transfusion transmission of both known and emerging pathogens. the technology uses a uv light source and riboflavin (vitamin b2) in combination to cause irreversible damage to nucleic acids (rna/dna). due to its mode of action, the riboflavin and uv process selectively renders viruses, bacteria and parasites, along with donor white cells, unable to replicate, while maintaining acceptable quality levels for platelets, red blood cells and plasma proteins [20] [21] [22] . beyond having proven effectiveness at reducing infectious titres in a variety of viruses, bacteria, and parasites [23] [24] [25] [26] [27] , the riboflavin and uv process has specifically demonstrated a high level of effectiveness against mers-cov [28] . the purpose of this study was to evaluate the efficacy of the mirasol prt system on the reduction of sars-cov-2 in both human plasma and platelet products. plasma products (n n = 5) whole blood products collected in cpd were acquired from an accredited blood bank after institutional review board (irb) approval and shipped to terumo bct. the whole blood products were held overnight at room temperature and then separated on an automated blood processing system to create pf24 plasma (plasma frozen within 24 h after phlebotomy to ≤-20°c). the units were stored at ≤-20°c until needed. leukoreduced apheresis platelet products suspended in plasma and collected in acd were acquired from an accredited blood bank under an irb-approved protocol and shipped to terumo bct. platelet products were allowed to rest for minimum of 2 h before being placed onto a platelet incubator/ shaker at 22°c -2. prior to use platelets products were evaluated for positive swirl and to ensure the incoming cell count was within 800-2100 x 10 3 /ll. the riboflavin and uv light process has been previously described in detail [24, 27] . briefly, 200 ml -5 of either plasma or platelets was dispensed into an extended life platelet (elp) illumination/storage bags (terumo bct, lakewood, co, usa) and then mixed with 35 ml of riboflavin solution (500 µmol/l riboflavin in 0á9% sodium chloride, ph 4á0 to 5á0 [terumo bct, larne, ireland]). a fixed product volume was used to simplify the amount of virus required for each unit. after units were spiked with virus, they were placed into the mirasol illuminator (terumo bct) for uv treatment. the units were exposed to 6á24 j/ml of energy. sars-cov-2 (isolate usa-wa1/2020) was acquired through bei resources. the virus was propagated in vero cells (atcc #ccl-81, manassas, va, usa) that were cultured in dulbecco modified eagle medium with high glucose (milliporesigma, st. louis, mo, usa) and 2% fetal bovine serum (peak serum, wellington, co, usa). after clarification by centrifugation, the virus stock was supplemented to 10% with fetal bovine serum, frozen, and maintained at -80°c until thawed for use. the virus reduction studies were performed at colorado state university by staff trained to operate the mirasol illuminator. all treatments occurred within a biosafety level 3 laboratory. for each 200 ml unit of either pf24 plasma or apheresis platelets containing riboflavin, a total of 12 ml of sars-cov-2 virus in media was added. after mixing, a pretreatment sample was obtained and held at ambient temperature until the completion of the treatment (<10 min). following treatment with riboflavin and uv light, a post-treatment sample was collected. samples were frozen and stored at -80°c until the plaque assay could be performed. each of the pretreatment samples was serially diluted from 10 -1 to 10 -7 in sterile phosphate-buffered saline (pbs), and the dilutions were plated in duplicate by plaque assay on vero cells. each of the post-treatment samples was serially diluted from 10 -0 to 10 -2 in pbs, and 6 replicate wells were plated for each dilution. the number of replicate wells in the post-treatment samples was increased to provide greater assay sensitivity to detect low levels of virus, if present. to perform the plaque assay, confluent vero cell monolayers were grown in 6-well tissue culture plates and each well was inoculated with 0á3 ml of the appropriate diluted sample. the plates were rocked every 10-15 min for 45 min and then overlaid with 0á5% agarose (life science products, frederick, co, usa) in media and incubated at 37°c, 5% co 2 . after 2 days, a second overlay containing 0á005% neutral red (mp biomedicals, irvine, ca, usa) was added and the plaques were counted the following day. the virus titre was determined based on the total plaque count and then corrected for dilution and the volume plated. when no virus is detected in the post-treatment samples at the lowest dilution tested, the limit of detection for the assay has been reached [29] . all of the values at the limit of detection were considered less than or equal to the calculated limit of detection. the theoretical limit of detection was calculated using the following equations: where n is the lowest number of particles in the product that can be detected with 1-p confidence; p is the probability that a virus will be undetected (95% confidence of detecting a virus, p = 0á05); v is the total volume of the treated product (platelet or plasma + riboflavin + virus); and v is the volume used for viral enumeration (volume inoculated per well in ml) 9 (number of replicate wells) 9 (lowest dilution inoculated). a total of five (n = 5) pf24 plasma units were evaluated using riboflavin and uv light against sars-cov-2. the average starting titre of the five units was 4á62 log pfu/ ml, and the mean log reduction was ≥3á40 log (table 1) . all five treated units were reduced to the limits of detection (≤1á22 log pfu/ml). the measured titre of the viral inoculum was 6á04 log pfu/ml, and the theoretical pretreatment titre based on the measured stock titre is 4á73 log pfu/ml. the theoretical pretreatment titre was in agreement with the average measured starting titre indicating that there was no neutralization of the virus by native immune components in the plasma products, nor was there an effect of sample handling on the measured viral titre. a follow-up study in platelets was also performed. three apheresis platelet products were inoculated with stock virus and treated with the riboflavin and uv process. the average pretreatment titre was 4á77 log pfu/ml, and the mean log reduction was ≥4á53 log ( table 2) . as with the plasma study, the virus titre was reduced to the limit of detection (≤0á25 log pfu/ml) in all three donor platelet units. the measured titre of the viral inoculum was 6á11 log pfu/ml, and the theoretical pretreatment titre based on the measured stock titre is 4á80 log pfu/ ml. again, the theoretical pretreatment titre was in agreement with the average measured starting titre indicating that sample handling did not affect the measured viral titre, nor was there any indication that native immune components in the platelet products neutralized the virus. the overall log reduction observed in both studies was limited by the titre of the viral stock. in the plasma study, the log reduction was further limited due to the 10 0 dilution exhibiting cytotoxicity in the post-treatment samples. this dilution was not included in the limit of detection calculation for the plasma study. additionally, one well of the six wells plated at 10 0 for the post-treatment sample in unit #2 of the platelet study was contaminated. this well was removed from the study, and this is reflected in the post-treatment titre calculation for this unit. while there have been no documented cases of transfusion transmission of sars-cov-2 to date [19], the pandemic this virus is causing illustrates the precarious nature of blood safety and its reliance on donor selection (donor questionnaire and health evaluation) and donor testing to prevent transfusion transmission of infectious diseases. undoubtedly, these two approaches have prevented countless numbers of transfusion transmitted diseases; however, the covid-19 pandemic has reinforced known weaknesses to the current blood safety strategy. during a pandemic, widespread donor deferrals can create both regional and national blood shortages, large populations of asymptomatic viremic donors may significantly increase the risk of transfusion transmitted diseases, and testing needs may outpace the ability of the marketplace to supply tests leaving blood units potentially unscreened for an outbreak agent. to combat potential blood shortages caused by the covid-19 pandemic, the fda has recently issued revised guidance documents easing traditional donor deferral periods, for example for certain malaria exposures, from 12 to 3 months [30,31]. however, this emergency response potentially brings in new donors who may add a risk of transfusion-transmissible infectious diseases given these donors may not be as well characterized as routine donors [32] [33] [34] . the implementation of an effective pathogen inactivation (pi) method can alleviate some of the above risks by adding in a proactive layer of blood safety that could lessen the need for widespread deferrals, reduce infectious titres in asymptomatic donors and provide time for screening tests to be approved and delivered to the market. based upon early case reports of positive clinical outcomes when used in conjunction with supportive care and anti-viral therapy, there has been significant interest in using covid-19 convalescent plasma as a therapeutic for infected patients [35] [36] [37] [38] . convalescent plasma is drawn from recovered patients, who might also be firsttime donors or come from a high-risk population, thus again collecting products from these donors may come with additional risk. including pi may be regarded as a prudent safety measure for convalescent plasma, particularly where it is to be used prophylactically for at-risk populations such as healthcare workers, first responders and those potentially exposed in localized outbreaks (nursing homes, ocean vessels, homeless shelters, incarceration facilities, etc.). pi would help guard against the residual risk of co-infections as well as a possible superinfection with the pandemic agent [39] . importantly, the available evidence also suggests that pi does not have a deleterious effect on antibody function [40, 41] . overall, pi methods have the ability to provide a proactive layer of blood safety through their broad-based effectiveness against a wide range of known pathogens [42] , as well as potential efficacy against unknown pathogens. during the first few months of a large outbreak or pandemic, they can provide a crucial first line of defence against transfusion transmission of an outbreaking agent along with reducing residual risk of co-infections when using convalescent plasma to treat both infected patients and at-risk populations. it should be noted though that pi methods also come with their limitations including that a donor's peak viremia may exceed a pi method's reduction capacity, that blood product types and specifications that can be treated with a given pi system may be limited, and that no technology has been shown to be universally successful against all classes of pathogens. however, in the midst of a pandemic or large-scale outbreak and a potential worsening blood supply situation, pi could play a vital role in maintaining a safe blood supply. riboflavin and uv light effectively reduced the titre of sars-cov-2 in both human platelet and plasma products to below the limit of detection using an in vitro plaque assay. these data complements previously collected data with mers, which was also reduced to below the limit of detection, and show this technology is effective against multiple coronavirus species. although the risk of transfusion transmission for sars-cov-2 is suspected to be low, implementing a pi technology like riboflavin and uv light may provide a crucial first line of defence against a future rapidly spreading agent that could theoretically be transmitted via blood transfusions. a novel coronavirus from patients with pneumonia in china outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle coronaviridae study group of the international committee on taxonomy of v: the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 sars and mers: recent insights into emerging coronaviruses recently discovered human coronaviruses sars-cov-2: an emerging coronavirus that causes a global threat a pneumonia outbreak associated with a new coronavirus of probable bat origin presumed asymptomatic carrier transmission of covid-19 protecting health-care workers from subclinical coronavirus infection viral attacks on the blood supply: the impact of severe acute respiratory syndrome in beijing stramer sl: current perspectives in transfusion-transmitted infectious diseases: emerging and re-emerging infections identification of a novel coronavirus in patients with severe acute respiratory syndrome quantitative analysis and prognostic implication of sars coronavirus rna in the plasma and serum of patients with severe acute respiratory syndrome detection of sars coronavirus in plasma by real-time rt-pcr viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection clinical features of patients infected with 2019 novel coronavirus in wuhan, china molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes 19 fda: updated information for blood establishments regarding the novel coronavirus outbreak plasma constituent integrity in pre-storage vs. post-storage riboflavin and uv-light treatment-a comparative study pathogen reduction of fresh plasma using riboflavin and ultraviolet light: effects on plasma coagulation proteins protein quality in mirasol pathogen reduction technology-treated, apheresis-derived freshfrozen plasma treatment of blood with a pathogen reduction technology using ultraviolet light and riboflavin inactivates ebola virus in vitro inactivation of viruses in platelet and plasma products using a riboflavinand-uv-based photochemical treatment treatment of platelet products with riboflavin and uv light: effectiveness against high titer bacterial contamination inactivation of plasmodium spp. in plasma and platelet concentrates using riboflavin and ultraviolet light photochemical inactivation of selected viruses and bacteria in platelet concentrates using riboflavin and light inactivation of middle east respiratory syndrome coronavirus (mers-cov) in plasma products using a riboflavinbased and ultraviolet light-based photochemical treatment cder, cber, 1998 30 fda: guidance for industry -revised recommendations for reducing the risk of human immunodeficiency virus transmission by blood and blood products. silver spring, md: ocod, 2020 31 fda: guidance for industry -revised recommendations to reduce the risk of transfusion-transmitted malaria trends in incidence and prevalence of major transfusion-transmissible viral infections in us blood donors risk factors for retrovirus and hepatitis virus infections in accepted blood donors development of a multisystem surveillance database for transfusion-transmitted infections among blood donors in the united states use of convalescent plasma therapy in two covid-19 patients with acute respiratory distress syndrome in korea effectiveness of convalescent plasma therapy in severe covid-19 patients treatment of 5 critically ill patients with covid-19 with convalescent plasma fda: recommendations for investigational covid-19 convalescent plasma. silver spring points to consider in the preparation and transfusion of covid-19 convalescent plasma treatment of blood with a pathogen reduction technology using ultraviolet light and riboflavin inactivates e bola virus in vitro characterization of ebola convalescent plasma donor immune response and psoralen treated plasma in the united states component pathogen inactivation: a critical review we thank bei for providing the sars-cov-2 utilized in these studies. the reagent was deposited by the centers for disease control and prevention and obtained through bei resources, niaid, nih: sars-related coronavirus 2, isolate usa-wa1/2020, nr-52281. the authors would also like to thank dr. lou katz for his critical review of this manuscript. this study was sponsored by terumo bct. this manuscript template is based on the international committee of medical journal editors (icmje) recommendations dated december 2014. the icmje recommends that authorship be based on the following 4 criteria: (1) substantial contributions to the conception or design of the work; or the acquisition, analysis or interpretation of data for the work; (2) drafting the work or revising it critically for important intellectual content; (3) final approval of the version to be published; (4) agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. key: cord-277811-j58qvyum authors: mehrani, hossein; ghanei, mostafa; aslani, jafar; tabatabaei, zahra title: plasma proteomic profile of sulfur mustard exposed lung diseases patients using 2-dimensional gel electrophoresis date: 2011-01-07 journal: clin proteomics doi: 10.1186/1559-0275-8-2 sha: doc_id: 277811 cord_uid: j58qvyum introduction: sulfur mustard "bis (2-chlroethyl) sulphide" (sm) is a chemical warfare agent that remains a threat to human health. the aim of this study was to identify protein expression signature or biomarkers that reflect chronic lung damages induced by sm exposure. methods: prior to analysis, plasma was fractionated using ethanol precipitation. using two dimensional sds-page; fractionated protein profiles of 20 healthy and 20 exposed patients with lung diseases were established. selected protein spots were successfully identified with maldi tof ms/ms. results: the results show that α1 haptoglobin isoforms were detected in plasma of the all lung disease patients but none of the healthy controls. amyloid a1 isoforms was also detected in plasma of the lung disease patients but none of the healthy controls. moreover, low molecular weight proteins were enriched in ethanol supernatant compared to ethanol precipitate. conclusion: our present results and previous studies suggest that ongoing tissue remodeling is involved in sm exposed lung damage patients. these finding might improve patient care and suitable therapies. sulfur mustard is a chemical warfare agent that remains a threat to human health.. more than lethality, sm causes debilitating effects that can leave an exposed individual incapacitated for days, months, or years. lung injury is a common health problem after inhalation, which leads to chronic bronchitis and interstitial lung diseases [1] . the clinical picture of the poisoning is well known from the thousands of victims during world war i and the recent iran-iraq conflict. in the latter, sulfur mustard was heavily used and at the present time about 30,000 victims still suffer from late effects of the agent, such as chronic obstructive lung disease, lung fibrosis, recurrent corneal ulcer disease, and chronic conjunctivitis [2] . late complications of mustard gas exposure and main clinical findings include; chronic bronchitis, bronchiectasis and bronchiolitis obliterans (bo) [3] [4] [5] . however, clinical manifestation in lung disorders due to sulfur mustard is different from other lung diseases, due to the fact that mustard lung is not responsive to corticosteroids. there is no common consensus about the pathophysiological basis of chronic pulmonary disease caused by this chemical warfare agent [6] . proteomics technologies can identify and quantify novel proteins in the plasma that can function as biomarkers of the presence or severity of disease states. in general, human plasma proteome profiling is challenging. albumin is present at about 40 mg/ ml and several other proteins are highly abundant including immunoglobulins (iggs), transferrin and fibrinogen which typically constitute greater than 90% of total protein mass [7] . these abundant proteins may hinder the detection of low-abundant proteins that can be of specific interest in the search for biomarkers of disease [8] . however, it is the low abundant proteins that are most likely to be biologically relevant as the markers of a disease state. for analysis of low-abundant proteins in plasma, many strategies have been developed for the selective removal of albumin and other highabundance proteins. albumin can be removed by immune affinity columns chromatography [9] , isoelectric trapping [10] , heparin chromatography [11] and peptide affinity chromatography [12] . however, it is well known that albumin and other high-abundance proteins may also act as carrier or transport proteins and thus are likely to bind many species of interest, such as peptide hormones, cytokines, and chemokines. there are wide-ranging interests in using the proteomics approach to define markers of lung disease. although respiratory tract lesions represent the major disability after sm exposure, only a few studies have investigated the long term pathophysiology of sm induced respiratory damages, in particular their proteomes. we have recently examined the proteomics pattern in bronchoalveolar lavage (bal) fluid of sm exposed patients and identified families of proteins whose expression is up or down regulated compared to healthy controls [13] . plasma proteins and peptides are from almost every tissue and cell, and their change in quantity and quality is specific not only to the tissue affected by disease, but also to the disease process itself. in addition, plasma is the most easily accessible, less invasive, and widely collected sample. we attempted to explore plasma proteomics patterns of these patients, using ethanol fractionation. tow-dimensional gel electrophoresis was applied and followed by maldi-tof ms to look for new markers in the plasma of exposed patients which may help in further understanding the nature of long term effects of mustard gas. these finding might improve patient care and finding suitable therapies. the plasma protein content of the patients and the controls are presented in table 1 . no significant differences were observed in plasma protein contents of patients and controls. the ethanol fractionation was used to enrich low molecular weight proteins. as shown in figure 1 , most of the low molecular weight proteins were enriched in the ethanol supernatant rather than the precipitate. we found that 50% (v/v) ethanol was more efficient in fractionating low molecular weight proteins. to avoid any protein losses from the sample, we used both supernatants and precipitates of these fractions for 2-de analysis. for the first dimension 24 cm ipg strips with ph values in the range of 4-7 were used. the proteins were resolved in homogeneous 15% acrylamide gels in the second dimension to obtain greater resolution for small proteins. about 300 spots in each colloidal cbb-stained gel can be visualized by imagemaster software. representative 2-de images of plasma profiles from a healthy control for 50% (v/v) ethanol precipitate and ethanol supernatant are shown in figure 2a and 2b respectively. comparing the proteomics patterns of these two gels shows that both immunoglobulin heavy and light chains are separated in ethanol precipitate ( figure 2a ) and albumin is distributed both in ethanol supernatant and precipitate (figure 2a and 2b) . moreover, most of the small molecular weight proteins in the range of 15-50 kda, have significantly (p < 0.05) higher spot volume and intensity in ethanol supernatant rather than precipitate ( table 2 and figures. 2a and 2a) . we analyzed the differences in the plasma protein patterns, comparing the gels of the diseased and healthy controls. the analysis of protein patterns of the plasma was focused on those protein spots which showed differences, comparing the patients and the controls. they were compared with image master 2-de software and indicated only protein results in all cases (100%) with the same condition. as shown in figures 2 b and 2c, twenty six protein spots were subjected to maldi tof ms analysis. all selected proteins and their isoforms were subsequently identified by pmf and ms/ms u s25% p25% s50% p50% s75% p75% figure 1 sds-page of plasma proteins fractionated with different concentrations (v/v) of ethanol. u represents plasma proteins before precipitation; s, ethanol supernatant; p, ethanol precipitate. samples were analysed as described in the method section and gels were stained using silver staining. analysis. table 3 lists the identities of the proteins and their isoforms which were analyzed in this experiment using maldi tof ms. figures 2b and 2c shows the location of these protein spots in the 2-de gels of a healthy controls and an exposed patients respectively. volume and intensity of those protein spots which were only present in all patients' plasma but none of the healthy controls are shown in table 4 . images from other healthy volunteers and patients were similar (data not shown). haptoglobin α1 chain isoforms (spots 21, 22 and 23) were only detected in the plasma of the severe lung diseases patients but were not detectable in healthy controls ( figure 2b and 2c). furthermore, serum amyloid a1 isoforms (spots 24 and 26) were only seen in the plasma of the patients but none of the healthy controls ( figure 2b and 2c). in this study we present plasma proteome analysis of sm exposed patients compared to the healthy controls. human plasma and serum represent important biological materials for disease diagnosis. however, the wide dynamic range in protein concentrations remains a major challenge in the development of diagnostic assays. human plasma albumin and the various forms of immunoglobulin represent the most abundant proteins in the plasma, constituting up to 80% of the total plasma proteins. the table 3 . classical depletion strategy for albumin involves using hydrophobic dye cibacron blue, a chlorotriazine dye which has high affinity for albumin [14] . as a group, the immunoglobulins represent the second most abundant proteins in the plasma or serum. it has been reported that albumin depletion may also remove some small low copy number proteins [15] . therefore, in these experiments we used ethanol fractionation and found that this simple and low cost experimental procedure can be used for removing immunoglobulins and part of the albumin. moreover, using 50% (v/v) fractionation, we showed that the ethanol supernatant contains all the protein spots that were found in the ethanol precipitate and recovers more small mw proteins. we have identified some proteins that could give a novel insight into the pathogenesis of mustard lung. one of the main findings was different isoforms of haptoglobin in the plasma of severe lung disease patients but not in healthy controls. haptoglobin is present in normal human plasma at a concentration range of 0.3-1.9 mg/ml, accounting for 0.4-2.6% of total plasma protein. human haptoglobin is an inflammation-inducible plasma protein. it consists of 2 different types of α chains and a single type of β chain connected by disulfide bridges (β-α-α-β) giving 3 major phenotypes (hp 1-1, hp 2-1, hp 2-2), the numbers 1 and 2 representing α1 (8.9 kda) and α2 (16 kda) chains, respectively. the β chain (40 kda) is heavier than α chain and is identical in all hp types [16] [17] [18] [19] . our results ( figures 2b and 2c) show that under our experimental conditions, the α2 chain (spots 11, 12, 13) is similarly expressed in the plasma of both experimental data are mean ± sem of the % volume and intensity of protein spots on the 2d gels multiplied by 100. significant difference between the samples were calculated using the student's t-test and marked by * if p < 0.05, ** if p < 0.01. a. spot numbers correspond to those in figures 2a and 2b groups, but α1 chain (spots 21, 22, 23) is only expressed in the patients plasma samples but not in the healthy controls. in our recent study of bal fluid proteomics patterns in sm exposed patients we also found that haptoglobin isoforms were significantly elevated in moderate and severe lung disease patients compared to mild and healthy controls [13] . acute-phase proteins are induced shortly after exposure to triggering events such as inflammation, infection, and trauma, and are thought to be part of a general defense-response in injured tissue. but, these patients have been exposed to sm more than 20 years ago. it seems that an ongoing damage is occurring in lung of these patients. a recent proteomic study has also shown that levels of haptoglobin are elevated in bal fluid in patients with mild asthma, and reported that haptoglobin may play a role in the differentiation of fibroblast progenitor cells, suggesting a novel role for haptoglobin in airway remodeling in patients with asthma [20] . conversely nishioka et al. showed that the serum concentration of haptoglobin was decreased during acute exacerbation in asthmatic children [21] . haptoglobin isoforms and fragments are also elevated in plasma of severe acute respiratory syndrome (sars) patients [22] . haptoglobin plays a crucial role in defense against hemoglobin-induced oxidative stress by a mechanism thought to involve its high-affinity binding with hemoglobin and preventing iron release from hemoglobin. however, it has yet to be shown that haptoglobin itself is an antioxidant molecule [23] . arredouani et al. [24] further described the role of haptoglobin affecting the immune system, showing that haptoglobin directly affects t-cells and suppress t-helper-cells through down-regulation of cytokine production. we found that amyloid a1 isoforms only detected in the plasma of severe lung diseases patients but not in healthy controls ( figure 2b and 2c) . similar results were reported for sars patients [22] . although serum amyloid a1 is not as commonly used in human medicine as c-reactive protein (crp), it is more sensitive to acute response than crp [25, 26] . serum amyloid a1 is an acute-phase protein that is induced, like crp, by inflammatory mediators, including il-6, il-1β, and tnf-α, that rises in acute exacerbation of copd [27] . serum amyloid a1 is secreted from the liver as the predominant apolipoprotein associated with plasma high density lipoprotein. we postulate that in our sm exposed patients ongoing tissue damage and repair (remodeling) is occurring which leads to an increase in acute phase reactant proteins such as haptoglobin and amyloid a1. in conclusion, this study complements our previous bal fluid proteome analysis of patients exposed to sm gas which resulted in identification of number of differentially expressed proteins. to our knowledge this is the first study of plasma fluid proteome in sm exposed subjects. in this and our previous study the patterns of differentially expressed proteins identified in sm exposed patients are somewhat different from other lung diseases. it seems that sm exposed lung has an aberrant tissue remodeling which results in abnormal tissue architecture. all chemicals used in these studies were analytical grade or equivalent and were obtained from sigma (st. louis, mo) unless otherwise noted. milli q water (18.2 mω) was used throughout. according to the american thoracic society (ats) classification and based on our spirometric and high resolution computed tomography (hrct) findings, the patients were classified as severe lung diseases condition. patients group included 20 male subjects. a group of 20 healthy age-matched male individuals was used as the control. this study was approved by the ethics committee of the research center of baqiyatallah university of medical sciences, and informed consent was obtained from all patients and healthy controls. all participants were free to leave the study at will. the participant patients were suffering from pulmonary disorders due to previous exposure to a single high dose of sm gas during the iran-iraq conflict in 1987. inclusion criteria were as follows: documented exposure to sm and documented diagnosis of chronic pulmonary disease due to mustard gas. exclusion criteria for the patients and the control subjects were history of a chronic disease (tuberculosis, diabetes, hypertension, heart disease, hepatic diseases, etc.), resection of one or more lobes of the lungs, pneumonia and/or acute bronchitis, cigarette smoking or substance abuse. none of the patients or control subjects had a history of allergy or asthma. all patients and controls were in a stable condition and none of the participants had been administrated corticosteroids during the two-month period immediately preceding the studies. fasting venous blood samples were collected in the morning (8-10 am). blood samples were drawn into tubes containing k2edta and then immediately centrifuged at 1300 × g and 4°c for 10 min according to hupo plasma proteomics project recommendation [28] . supernatant was removed to a new tube and one tablet of complete protease inhibitor cocktail (roche, mannheim, and germany) was added. then, sample were promptly frozen in aliquots and stored at -80°c until used. plasma samples were thawed at room temperature and centrifuged at 10,000 × g for 10 min at 4°c. then clear supernatants were transferred to new microfuge tubes for further processing. total protein in plasma fluid was determined by the bicinchoninic acid (bca) assay and employed bovine albumin as the standard (pierce, rockford, il). the concentrations of proteins in plasma and different fractions were determined using a standard curve generated by the absorbance at 562 nm. following procedures for ethanol fractionation were applied. all steps were performed at 4°c. plasma samples were diluted 1:1 (v/v) with milli q water in a new microfuge tube and equilibrated to 4°c by gentle mixing (continuously on a vortex mixer at a low setting) for 10 min. in new separate microfuge tubes containing 500 μl of diluted plasma samples, different volumes of cold ethanol were added and total volume was adjusted to 1000 μl with milli q water. samples were incubated for an additional hour with gentle mixing on the vortex mixer and were centrifuged at 10,000 × g for 15 min at 4°c. supernatants were removed to new tubes, pellets were briefly re-centrifuged and any residual supernatant was removed and added to the previous supernatant fraction. for electrophoresis, the supernatants were dialyzed against 50 mm tris-hcl (ph 7.4) overnight to remove ethanol. for one dimensional sds-page analysis the collected ethanol supernatants and pellets were reconstituted in 50 mm tris-hcl (ph 6.8), 2% (w/v) sds, 0.1% (w/v) bromophenol blue and 10% (v/v) glycerol. proteins were separated by mini 1-de (10% t resolving gels with 4% stacking gels; 10 μg protein/lane). gels were electrophoresed at 30 v for 30 min and then at 50 ma per gel for 45 min, and stained using vorum silver staining procedure as described recently [29] . for 2-de analysis dialyzed ethanol supernatants and pellets were reconstituted in buffer consisting of 7 m urea, 1 m thiourea, 20 mm tris, ph 7.5, 4% (w/v) 3-[(cholamidopropyl) dimethylamino]-1-propanesulfonate (chaps), and centrifuged at 10,000 × g at room temperature for 5 min. supernatants were taken for 2-de gel analysis. protein concentration in the recovered samples was determined using a modification of the method as described by bradford [30] . two dimensional gels were analysed using 400 μg proteins per 24-cm linear ipg strips ph 4-7 (bio rad) as described previously [13] . ipg strips were reduced and alkylated, and then proteins were separated in the second dimension on homogeneous 15% polyacrylamide gels in an ettan daltsix electrophoresis unit (ge healthcare, uppsala, sweden). ipg strips were placed on the top of polyacrylamide gels (1 × 200 × 260 mm) and sealed with a solution of 1% (w/v) agarose containing a trace of bromophenol blue. gels were run in a running buffer, containing 25 mm tris, 192 mm glycine, and 0.1% (w/v) sds at 2w/gel for 45 min, followed by 6w/gel at 20°c until the bromophenol blue had migrated to the bottom of the gel. gels were fixed overnight in acetic acid, methanol, water, 10:45:45 (v/v) and stained with colloidal coomassie brilliant blue (cbb) g-250 as described previously [13] . analysis of spot patterns was performed using imagemaster 2d platinum software (version 6.0; ge healthcare). cbb stained gels were scanned in transmission scan mode using bio-rad gs-800 calibrated densitometer at a resolution of 600 dots per square inch (dpi). the scanned gels were saved as tif images for subsequent analysis. protein spots were detected automatically. manual spot editing or deleting (of artifacts) was performed when necessary. the spots intensity, volume, or saliency was adjusted in the preview mode using image master software. furthermore spots on all gels were visually carefully inspected for inappropriate matching, staining artifacts, or bad spot detection and conserved for analysis. to measure the volume and intensity of protein spots on cbb-stained gels the volume and intensity of each spot was divided by the total volume or intensity of all spots of the same gel. since this method of normalization produces extremely small values, the result was multiplied by a scaling factor of 100, which produced spot percentage volumes or intensity. only those spots that were reproducibly and statistically significant (p < 0.05) in intensity or volume were used for analysis. for mass analysis the protein spots of interest were aseptically removed under a laminar flow hood. processing of gel plugs, trypsin digestion and maldi tof ms/ms analysis using a bruker autoflex iii maldi tof/tof instrument (alphalyse, denmark) and database searching was performed as described previously [13] . the ms and ms/ms spectra were combined and used for a database search using the mascot software (matrix science, version 2.2.03) for database searches with the selection of following criteria: database search program: nrdb1 (6655203 protein sequence), species of origin homo sapience, peptide ion mass tolerance 60 ppm,, ms/ms tolerance 0.2 da, peptide cut-off of 25, and digestion by trypsin allowing for no more than one missed cleavage. the accuracy of mass detection was mh+ of 0.01 assuming possibility of modification of cysteine by acrylamide and oxidation of methionine. proteins identification was based on a combination of the peptide mass fingerprint (pmf) with peptide masses, and several ms/ms spectra of selected peptides in each maldi ms spectrum. the protein and protein isoforms shown on the identification list is based purely on the human protein database accession that gives the highest mascot score. to the extent that the different sequence isoforms are represented in individual database accession numbers, these have been considered in the database search. other modifications and isoforms are not considered in this analysis. the positive protein identification was based on a probability-scoring algorithm (http://www.matrixscience. com) and the 95% confidence level for positive identification was a score = 80 all data are representative of at least three independent experiments with twenty individuals in each group and are expressed as means ± standard error of the mean (sem). comparisons between two groups were performed using unpaired student's t-test. the criterion for statistical significance was p < 0.05 for all comparisons. tracheobronchomalacia and air trapping after mustard gas exposure medical aspects of sulfur mustard poisoning the diversity of the effects of sulfur mustard gas inhalation on respiratory system 10 years after a single, heavy exposure: analysis of 197 cases bronchiolitis obliterans in a survivor of a chemical weapons attack an international collaborative pathologic study of surgical lung biopsies from mustard gas-exposed patients long term consequences from exposure to sulfur mustard: a review the human plasma proteome: history, character and diagnostic prospects efficient prefractionation of low-abundance proteins in human plasma and construction of a two-dimensional map sample preparation of human serum for the analysis of tumor markers. comparison of different approaches for albumin and gamma-globulin depletion depletion of the highly abundant protein albumin from human plasma using the gradiflow heparin chromatography to deplete high-abundance proteins for serum proteomics development of mammalian serum albumin affinity purification media by peptide phage display bronchoalveolar lavage fluid proteomic patterns of sulfur mustardexposed patients studies on the mechanism of binding of serum albumin to immobilized cibacron blue f3ga albumin depletion of human plasma also removes low abundance proteins including the cytokines differentially expressed serum haptoglobin alpha chain isoforms with potential application for diagnosis of head and neck cancer covalent structure of human haptoglobin: a serine protease homolog gene action in the human haptoglobins. 3. isolation of the α-chains as single gene products. isolation, molecular weight, and amino acid composition of α-and β-chains hplc analysis of discrete haptoglobin isoform n-linked oligosaccharides following 2d-page isolation specific haptoglobin expression in bronchoalveolar lavage during differentiation of circulating fibroblast progenitor cells in mild asthma alpha-1-antitrypsin and complement component c7 are involved in asthma exacerbation plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry antioxidant role of human haptoglobin haptoglobin directly affects t cells and suppresses t helper cell type 2 cytokine releases serum amyloid a is a biomarker of acute exacerbations of chronic obstructive pulmonary disease the association of sensitive systemic inflammation markers with bronchial asthma copd exacerbations. 2: etiology. thorax spirometric classification hupo plasma proteome project specimen collection and handling: towards the standardization of parameters for plasma proteome samples a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding plasma proteomic profile of sulfur mustard exposed lung diseases patients using 2-dimensional gel electrophoresis the authors are grateful to baqiyatallah university research branch and chemical injuries research center for a supporting grant. we also thank dr. ejvind mørtz in alphalyse, denmark for mass analysis. authors' contributions hm carried out study design, analysis of proteomics profile, data mining and drafted and managed the manuscript. mg participated in the design of study and patient handling. ja participated in the patient handling and funding management. zt participated in the experimental laboratory work on proteomic profiling. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-304616-k92fa15l authors: izes, aaron m.; kimble, benjamin; norris, jacqueline m.; govendir, merran title: assay validation and determination of in vitro binding of mefloquine to plasma proteins from clinically normal and fip-affected cats date: 2020-08-05 journal: plos one doi: 10.1371/journal.pone.0236754 sha: doc_id: 304616 cord_uid: k92fa15l the antimalarial agent mefloquine is currently being investigated for its potential to inhibit feline coronavirus and feline calicivirus infections. a simple, high pressure liquid chromatography assay was developed to detect mefloquine plasma concentrations in feline plasma. the assay’s lower limit of quantification was 250 ng/ml. the mean ± standard deviation intraand inter-day precision expressed as coefficients of variation were 6.83 ± 1.75 and 5.33 ± 1.37%, respectively, whereas intraand inter-day accuracy expressed as a percentage of the bias were 11.40 ± 3.73 and 10.59 ± 3.88%, respectively. accordingly, this validated assay should prove valuable for future in vivo clinical trials of mefloquine as an antiviral agent against feline coronavirus and feline calicivirus. however, the proportion of mefloquine binding to feline plasma proteins has not been reported. the proportion of drug bound to plasma protein binding is an important concept when developing drug dosing regimens. as cats with feline infectious peritonitis (fip) demonstrate altered concentrations of plasma proteins, the proportion of mefloquine binding to plasma proteins in both clinically normal cats and fip-affected cats was also investigated. an in vitro method using rapid equilibrium dialysis demonstrated that mefloquine was highly plasma protein bound in both populations (on average > 99%). successful treatment of feline infectious peritonitis (fip), a fatal, virulent coronavirus infection affecting predominantly younger cats, remains difficult [1] . reviews describing the virus responsible for fip as well as issues with diagnostics and therapeutics are available [1, 2] , providing an explanation as to why re-purposing drugs used in human medicine has been largely unsuccessful in treating fip infected cats [1] . recently, the use of antiviral therapies to alter the replication of virulent forms of feline coronavirus known as feline infectious peritonitis virus (fipv) has shown enormous hope for clinicians [3, 4] . however, veterinary access to these antiviral treatments is currently limited, leading to the global emergence of expensive, unregistered versions of these drugs without the necessary quality control assurances [4, 5] . accordingly, the need for inexpensive, safe, antiviral medications to treat fipv-infected cats remains. plos one | https://doi.org/10.1371/journal.pone.0236754 august 5, 2020 1 / 10 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 mefloquine is currently used for both prevention (as a monotherapy) and treatment (either alone, or in combination with artesunate) of chloroquine-resistant plasmodium falciparum malaria in humans [6] [7] [8] . it also has been shown to reduce the viral load of fipv and feline calicivirus (fcv) at low concentrations in infected crandell rees feline kidney cells without cytotoxic effects [9, 10] . using an in vitro assay, our team demonstrated that while mefloquine undergoes some phase i metabolism in cats, it does not undertake phase ii glucuronidative metabolism in this species [11] . in anticipation of conducting in vivo clinical trials of mefloquine to treat fipv-infected cats, mefloquine's plasma concentrations must be monitored to optimise the dosage regimen. knowledge of the amount of drug bound to plasma proteins is also important when optimising dosage regimens as only the unbound (or free) fraction is therapeutically active [12, 13] . although the plasma protein binding of mefloquine has been determined to be > 98% in humans [14] , its binding percentage in cats has not been reported. this information is particularly germane in any investigation of a potential fip antiviral as fip-affected cats have altered concentrations of the plasma proteins albumin (decreased) and globulins (elevated) compared to clinically normal cats [1, 15] . consequently, the aim of this study was two-fold: first, to develop and validate a high pressure liquid chromatography (hplc) method to detect mefloquine in feline plasma, and second, to determine the in vitro plasma protein binding of mefloquine in both clinically normal and fip-affected cats. mefloquine hydrochloride and verapamil hydrochloride (as the internal standard [is]) were purchased from sigma-aldrich (castle hill, nsw, australia). hplc grade methanol, hplc grade acetonitrile, phosphate buffered saline ph 7.4 (pbs) and triethylamine were purchased from thermo fisher scientific (macquarie park, nsw, australia). mefloquine and verapamil were quantified in feline plasma based on modified hplc method [16] . the hplc system consisted of a shimadzu lc-20at delivery unit, dgu-20a3 ht degassing solvent delivery unit, sil-20a auto injector, spd-20a uv detector and cto-20a column oven. shimadzu lc solution software version 1.24 (kyoto, japan) was used for chromatographic control, data collection and data processing. chromatographic separation was performed with a microsorb-mv c18 column (250 mm × 4.6 mm i.d., 5.0 μm; varian, mulgrave, vic, australia) with a 1.0 mm optic-guard c18 pre-column (choice analytical, thornleigh, nsw, australia) under a pressure of 1,900 psi at 25˚c. the isocratic mobile phase consisted of a mixture of 25 mm sodium phosphate buffer with 1.0% triethylamine adjusted to ph 2.5 and acetonitrile and methanol (50:25:25, v/v/v) at a flow rate of 1.0 ml/min. the injection volume was 10 μl per sample. the diode array detector was set at a wavelength of 220 nm. the retention times of verapamil and mefloquine were approximately 8.5 and 14.5 minutes, respectively. stock solutions of mefloquine and verapamil were prepared in 100% methanol and 100% acetonitrile, respectively, both at a concentration of 1.0 mg/ml. further working solutions of mefloquine were prepared in 100% methanol to achieve final concentrations of 500, 1,000, 2,500, 5,000, 10,000, 25,000 and 50,000 ng/ml. similarly, working solutions of verapamil were prepared in 100% acetonitrile to yield final concentrations of 12.5 μg/ml for the validation study and 5.0 μg/ml for the plasma protein binding study. stock solutions and working solutions were stored at 4.0˚c prior to use. blank, clinically normal, feline plasma was pooled (n > 5) and stored at -20˚c prior to use for preparation of standards and quality control (qc) samples. the origin of the plasma samples is described below. mefloquine standard samples (50, 100, 250, 500, 1,000, 2,500 and 5,000 ng/ml) and three quality control samples (qc) (250, 1,000 and 5,000 ng/ml) were prepared by spiking 10 μl of prepared working solutions of mefloquine into 90 μl of blank feline plasma. the proteins within the plasma samples were extracted through simple protein precipitation. specifically, 100 μl of acetonitrile, containing either 12.5 μg/ml of the is for the validation study or 5.0 μg/ml of the is for the plasma protein binding study, was added to the 100 μl feline plasma samples. the samples were then vortexed and centrifuged at 14,000 × g for ten minutes to remove any particulates. the supernatant was injected into the hplc system. assay selectivity was established by analysing pooled (n > 5) blank, clinically normal, feline plasma to ensure that there were no endogenous interference peaks around the retention times of mefloquine and verapamil. mefloquine concentrations were measured via standard curves performed on three replicates of each concentration of mefloquine (100, 250, 500, 1,000, 2,500 and 5,000 ng/ml) on three consecutive days. a weighting factor (1/x) was used to assign the relative importance of the observations in the regression; in this case, to ensure that larger observations were not over-fitted. the theoretical lower limit of detection (llod, the lowest analyte concentration that can be distinguished from the assay background noise) and the lower limit of quantification (lloq, the lowest concentration at which an analyte can be reliably detected at a specified level of accuracy and precision) were estimated as follows [17] : llod = 3.3 x σ /s, where σ is the standard deviation of the y-intercepts from the regression lines and s is the mean slope of the calibration curves. thereafter, the lloq was calculated using the formula lloq = 3 x llod. acceptance criteria for the lloq was defined as precision with a cv � 15% and accuracy within ± 20% of nominal concentration with repeated analyses [18] . intra-and inter-day precision, expressed as cv (%), were analysed from triplicates of qc samples (250, 1,000 and 5,000 ng/ml), both within a single day and on three consecutive days, respectively. intra-and inter-day accuracy, expressed as bias, were determined by a percentage difference between the estimated value and the nominal value of mefloquine as follows: bias (%) = 100 -(estimated value / nominal value × 100). absolute recovery of mefloquine and is were determined by comparing the peak area of pre-spiked plasma samples (n = 9) at concentrations of 250, 1,000 and 5,000 ng/ml with corresponding concentrations of mefloquine and is in mobile phase. each assay was conducted in triplicate. the linearity of the data was assessed through linear regression analysis with graphpad prism software version 8.1.1 (graphpad software, inc., ca, usa). with owner verbal or written consent, blood was collected from both clinically normal and fip-affected cats. the use of the residual plasma from this patient for this study was approved by the university of sydney animal ethics committee (protocol: 2016/1027). the animals were considered clinically normal based on inclusion criteria modified from that of norris et al. (2007) [19] . specifically, the cats designated for inclusion as clinically normal subjects were systemically well and had their blood collected for purposes other than investigating a current illness. examples of clinically normal cats included those presenting for annual examinations, routine vaccinations or for routine screening tests prior to sedation or general anaesthesia for de-sexing, grooming, routine dental scaling or radiography post-acute trauma [19] . in contrast, a cat's fip status was confirmed by direct immunofluorescence of effusion samples and immunohistochemistry on tissue samples (n = 5), or simply immunohistochemistry (n = 5) using methods outlined by worthing et al. (2012) [20] . additionally, other haematological tests were performed including quantification of albumin: globulin which was consistent with the hyperglobulinaemia observed with cats infected with fipv [15] . all plasma samples were stored at -20˚c prior to analysis. plasma samples from both clinically normal and fip-affected cats were provided by the paddington cat hospital (sydney, nsw) and the veterinary pathology and diagnostic service, sydney school of veterinary science. the in vitro plasma protein binding (ppb) of mefloquine was determined by the rapid equilibrium dialysis (red) method. the red assay was performed according to manufacturer's (thermo fisher scientific, macquarie park) instructions. pooled plasma samples (n > 5) were assigned to one of two experimental groups, depending on health status, i.e., either clinically normal cat plasma or fip-affected plasma. prior to experimentation, the ph of the pooled plasma (approximately ph 8) was adjusted to ph 7.3 [21] , to mimic the physiologic ph of cats. likewise, the total protein (tp), albumin and globulin concentrations of the pooled plasma samples were quantified prior to the assay by the vpds using a thermo fisher scientific konelab prime 30i analyzer (scoresby, vic, australia) employing conventional protocols. for each experimental group, two final concentrations of mefloquine (either 5,000 or 10,000 ng/ml) were tested using three replicates for each concentration. these concentrations were selected as they are comparable to the 10 μm concentration (10 μm = 3.78 μg/ml or 3,780 ng/ml) tested by mcdonagh et al. (2014) [10] in fipv-infected crandell rees feline kidney cells. accordingly, 300 μl of plasma pre-spiked with mefloquine was added to the redringed chamber of the red device whilst 550 μl of pbs was added to the white-ringed chamber. the unit was covered with sealing tape (thermo fisher scientific, scoresby, vic, australia, product no. 15036) and incubated on an orbital shaker at 250 rpm for four hours. the temperature was set at 38˚c to mimic the normal body temperature of cats. following incubation, 100 μl of post-dialysis samples were removed from the red-ringed chambers. these samples were then placed in separate microcentrifuge tubes and precipitated using 100 μl of chilled acetonitrile pre-spiked with the is (5.0 μg/ml verapamil). after vortexing, the mixtures were centrifuged at 14,000 × g for ten minutes. the supernatant was injected into the hplc system. similarly, 200 μl volumes of post-dialysis samples were removed from the whiteringed chambers. these samples also underwent hplc analysis. all samples were prepared and analysed in triplicate. the ppb of mefloquine was determined by the following equation: %free ¼ ðconcentration buffer chamber=concentration plasma chamberþ � 100% %bound ¼ 100 à %free statistical data analysis was performed using graphpad prism software version 8.1.1 (graph pad software, inc., ca, usa). prior to parametric testing, a shapiro-wilk normality test performed on the mefloquine ppb data indicated a normal distribution. likewise, an inspection of a normal quantile-quantile (qq) plot of this data further corroborated its normality. a twoway anova evaluating the effects of health status and drug concentration on mefloquine ppb was performed. results were considered statistically significant at p < 0.05. based on the uv spectra, the greatest area under the mefloquine peak was at a wavelength of 220 nm. the retention times of the is and mefloquine were approximately 8.5 and 14.5 minutes, respectively. no endogenous interference was observed at the retention times of mefloquine and the is. chromatograms of feline plasma pre-spiked with mefloquine and the is as well as blank feline plasma are shown in fig 1. selectivity. pooled blank feline plasma and feline plasma pre-spiked with mefloquine (250, 1,000 and 5,000 ng/ml) and the is (12.5 μg/ml) were used to check the selectivity of this method. due to the limited volume of fip-affected feline plasma available, only blank plasma of clinically normal cats was used. as demonstrated in fig 1, no endogenous plasma components interfered with elution of analytes. linearity, llod, lloq, accuracy and precision. the plasma peak ratio (area of mefloquine divided by the is area) versus the concentration was plotted and determined to be linear for the concentration range used (100 to 5,000 ng/ml). the mean regression standard curves (n = 3) for mefloquine were described as y = 9.374 e-005 x + 0.001417, with a weighting factor of 1/x. the correlation coefficient value (r 2 ) for each curve � 0.97. estimated from standard curves, the llod for mefloquine was 20.5 ng/ml and the lloq was 61.5 ng/ml. however, as mefloquine concentrations < 250 ng/ml were not accurate (>20% accuracy), the lloq was set as 250 ng/ml. intra-and inter-day precision expressed as cvs ranged from 5.30 to 8.74% and 3.77 to 6.32%, respectively. intra-and inter-day accuracy expressed as a percentage of the bias ranged from -12.67 to 13.96% and -15.01 to 9.04%, respectively. these values satisfied the guidelines regarding assay reliability [18] . intra-and inter-day precision and accuracy are summarised in table 1 . drug recovery from plasma. the absolute recovery rates of mefloquine expressed as percentages ± standard deviation (s.d.) from the 250, 1,000, and 5,000 ng/ml qc samples (n = 3) were 107.82 ± 5.93; 99.91 ± 5.58; and 105.21 ± 6.32, respectively. the absolute recovery rate of the is expressed as a percentage ± s.d. was 123.74 ± 3.77 (n = 9). in vitro plasma protein binding of mefloquine in healthy and fip-affected cats. the measured concentrations of total protein, albumin and globulin in the pooled plasma for the respective experimental groups are provided in table 2 . although reference intervals (ri) vary depending on the laboratory and the methodology used for quantification, reference intervals from cornell university's animal health diagnostic center [22] were used as none were provided by the testing laboratory. the results of in vitro plasma protein binding of mefloquine in clinically normal and fipaffected cats are provided in table 3 . a significant difference was found between the plasma protein binding of mefloquine in clinically normal and fip-affected cats (p = 0.0004), even though mefloquine was determined to be highly plasma protein bound (on average > 99%) in both groups. moreover, a significant interaction effect between health status and mefloquine concentration was identified (p = 0.0007). in contrast, no significant difference was discovered between the plasma protein binding of the two concentrations of mefloquine (p = 0.11). there has been recent interest in the suitability of mefloquine as an antiviral treatment for fip [9, 10, 23] . the majority of publications that detect and quantify mefloquine use liquid chromatography. liquid chromatography demonstrates good sensitivity (i.e., it can detect low concentrations of mefloquine), is highly specific and the machinery is affordable for veterinary diagnostic laboratories. although an hplc assay to detect mefloquine in an in vitro matrix has been reported [23] , in contrast, this study describes an assay to quantify mefloquine table 1 . intra-and inter-day precision and accuracy for the qc samples (250, 1000 and 5000 ng/ml of mefloquine) tested in triplicate for each concentration. to the authors' knowledge, this is the first report of mefloquine ppb in a non-human species. this study provides the novel finding that mefloquine is highly plasma protein bound in cats. in humans, mefloquine is also highly plasma protein bound (> 98%) [14] , which may account, in part, for its long half-life of approximately three weeks in healthy human subjects [24] . high plasma protein binding can result in the drug-protein complex acting as a reservoir for the physiologically active free drug concentration and consequently prolonging its duration of action [12, 25] . likewise, it should not be assumed that a drugs' ppb is constant across species [25] . in a comparative species study investigating the plasma protein binding of 574 compounds, drugs tend to be slightly more bound in human plasma proteins in comparison to the plasma proteins of rats, mice and dogs [26] . some of the compounds that showed significant interspecies differences in plasma protein binding included diazepam (98% ppb in humans versus 84.8% in rats), prazosin (94.7% ppb in humans versus 65% in rats versus 63% in dogs) and sildenafil (93.6% ppb in humans versus 74% in dogs) [26] . diseases, such as fip, can alter the concentrations of plasma proteins and in turn impact upon a drug's plasma protein binding and ultimately its therapeutic efficacy [27] . for example, in humans, the proteins albumin and α 1 -acid glycoprotein (aag) provide the largest contribution to the protein binding of drugs [27, 28] . yet, with acute falciparum malaria, serum concentrations of aag in non-immune human patients increase two-fold within 24 hours whereas plasma levels of albumin decrease by 30% [29] . likewise, in fip-affected cats, common serum protein abnormalities may include elevated aag [30] and globulin levels [1, 15] as well as decreased albumin levels and a low a: g ratio [15] . here, as described in the literature, the confirmed fip plasma samples demonstrated elevated globulin and decreased albumin levels as well as a low a: g ratio. mefloquine has a high affinity for aag binding, preferentially binding to aag over albumin [27] . thus, if more aag is present in fip-affected cats, it may be preferentially bound by mefloquine. yet, small changes in the unbound drug fraction of highly protein bound drugs can have a significant therapeutic impact [28] . for example, the reduction of ppb from 99.9% to 99.8% can lead to a doubling of therapeutically active unbound drug concentration in plasma [28] . here, although a significant difference was found between the plasma protein binding of mefloquine in clinically normal and fip-affected cats, due to the unknown biological variability of the assay, it is likely that this difference is equivocal. fundamentally, it is difficult to generalise from two pooled plasma samples to two populations of individuals since the biological variability in each population is unknown. the variability (standard deviation) used to evaluate differences in ppb were derived from the assay method and do not represent biological differences. yet, it is also unlikely that further in vitro mefloquine feline plasma protein binding studies will resolve this issue. on the contrary, observing the clinical response of mefloquine in fip-affected animals ultimately may decide mefloquine's therapeutic efficacy in this patient population. in vitro studies to quantify plasma protein binding have some limitations. whilst the major advantage of the red method include its speed, simplicity, reliability and cost-effectiveness [31] , its drawbacks, which also serve as limitations to this study, involve nonspecific membrane binding of the drug [31] as well as changes in oncotic pressure leading to an overestimation of unbound drug concentration [32] . moreover, in vitro ppb may not mirror in vivo ppb in the live animal as the structure of the plasma proteins and their affinity to bind substrates can vary with age, disease and /or presence of competing endogenous and exogenous compounds such as dietary constituents or other therapeutic drugs [33, 34] . furthermore, the drug-protein dissociation rate, which can also greatly affect highly ppb drugs [35] , was not determined in this study. likewise, an additional limitation to this project was the scarcity of confirmed fipaffected plasma as this impacted upon its availability for use in both the hplc validation and ppb protocols. finally, given mefloquine's purported mechanism of action as a schizonticide, another potential limitation concerns the effects of the intraerythrocytic accumulation of mefloquine on plasma protein binding. however, previous studies have demonstrated that red blood cells do not serve as a significant depot for mefloquine [36, 37] . recently, the broad spectrum coronavirus protease inhibitor, gc376 [38] , and the adenosine nucleoside analogue, gs-441524 [3, 4] have been shown to be safe and efficacious antiviral agents for use against fipv. yet, as neither of these agents has obtained registration for veterinary use, investigations into more readily available drugs with anti-fipv activity, such as mefloquine, are urgently required for this invariably fatal disease. this study has validated an accurate and reliable assay to detect mefloquine in feline plasma and demonstrated that mefloquine is highly plasma protein bound in both clinically normal and fip-affected cats. further studies describing mefloquine's pharmacokinetic profile in the cat should be progressed. an update on feline infectious peritonitis: diagnostics and therapeutics a review of feline infectious peritonitis virus infection: 1963-2008 the nucleoside analog gs-441524 strongly inhibits feline infectious peritonitis (fip) virus in tissue culture and experimental cat infection studies efficacy and safety of the nucleoside analog gs-441524 for treatment of cats with naturally occurring feline infectious peritonitis fifty years' fascination with fip culminates in a promising new antiviral mefloquine for preventing malaria in pregnant women the position of mefloquine as a 21 st century malaria chemoprophylaxis patient age does not affect mefloquine concentrations in erythrocytes and plasma during the acute phase of falciparum malaria antiviral effect of mefloquine on feline calicivirus in vitro identification and characterisation of small molecule inhibitors of feline coronavirus replication in vitro hepatic metabolism of mefloquine using microsomes from cats, dogs and the common brush-tailed possum (trichosurus vulpecula) predicting plasma protein binding of drugs: a new approach drug-protein binding: a critical review of analytical tools clinical pharmacokinetics of mefloquine positive predictive value of albumin: globulin ratio for feline infectious peritonitis in a mid-western referral hospital population a high performance liquid chromatographic assay of mefloquine in saliva after a single oral dose in healthy adult africans defining limit of detection and limit of quantitation as applied to drug of abuse testing: striving for a consensus international conference on harmonisation of technical requirements for registration of pharmaceuticals for human use prevalence of feline immunodeficiency virus infection in domesticated and feral cats in eastern australia risk factors for feline infectious peritonitis in australian cats ph adjustment of human blood plasma prior to bioanalytical sample preparation chemistry (cobas) reference intervals in vitro hepatic metabolism of mefloquine using microsomes from cats, dogs and the common brush-tailed possum clinical application of mefloquine pharmacokinetics in the treatment of p. falciparum malaria in vitro binding of cefovecin to plasma proteins in australian marsupials and plasma concentrations of cefovecin following single subcutaneous administration to koalas (phascolarctos cinereus) species differences in drug plasma protein binding selective plasma protein binding of antimalarial drugs to α1-acid glycoprotein plasma protein binding and blood-free concentrations: which studies are needed to develop a drug? serum protein concentrations in plasmodium falciparum malaria critical assessment of the diagnostic value of feline α1-acid glycoprotein for feline infectious peritonitis using the likelihood ratios approach protein binding of antimicrobials: methods for quantification and for investigation of its impact on bacterial killing errors in estimating the unbound fraction of drugs due to the volume shift in equilibrium dialysis clinical pharmacology: plasma protein binding of drugs what is the true clinical significance of plasma protein binding displacement interactions? drug safety characterization of plasma protein binding dissociation with online spe-hplc characterization of in vivo metabolites of wr319691, a novel compound with activity against plasmodium falciparum studies of the disposition and metabolism of mefloquine hcl (wr 142,490), a quinolinemethanol antimalarial, in the rat reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor professor michael court (washington state university, college of veterinary medicine) provided invaluable insight into the interpretation of the mefloquine plasma protein binding results. we also thank the veterinary pathology and diagnostics services, sydney school of veterinary science and dr. randolph baral (the paddington cat hospital, sydney, nsw), for providing the feline plasma samples. key: cord-329228-yjvw2ee1 authors: shikata, n.; maki, y.; noguchi, y.; mori, m.; hanai, t.; takahashi, m.; okamoto, m. title: multi-layered network structure of amino acid (aa) metabolism characterized by each essential aa-deficient condition date: 2006-10-13 journal: amino acids doi: 10.1007/s00726-006-0412-0 sha: doc_id: 329228 cord_uid: yjvw2ee1 the concentrations of free amino acids in plasma change coordinately and their profiles show distinctive features in various physiological conditions; however, their behavior can not always be explained by the conventional flow-based metabolic pathway network. in this study, we have revealed the interrelatedness of the plasma amino acids and inferred their network structure with threshold-test analysis and multilevel-digraph analysis methods using the plasma samples of rats which are fed diet deficient in single essential amino acid. in the inferred network, we could draw some interesting interrelations between plasma amino acids as follows: 1) lysine is located at the top control level and has effects on almost all of the other plasma amino acids. 2) threonine plays a role in a hub in the network, which has direct links to the most number of other amino acids. 3) threonine and methionine are interrelated to each other and form a loop structure. the recent advances in experimental technology made it possible to analyze various kinds of metabolites in biological samples comprehensively. the amounts of the metabolites in biological fluids and tissues change temporally in coordination with physiological conditions in complex metabolic and signaling pathways. multivariate analysis and pattern recognition studies have revealed that these metabolite profiles contain the phenotypic information which can be used as a signature for a physiological condition (nicholson et al., 1999) . amino acids are a group of metabolites which are important as substrates for protein synthesis as well as signaling molecules (felig, 1975) . the plasma amino acid concentrations, like other metabolites, have distinctive features for various physiological conditions. some diseases such as liver failure (holm et al., 1999) , renal failure (hong et al., 1998) , cancer (watanabe et al., 1984) , diabetes (watanabe et al., 1983) , muscle dysfunction (jimenez jimenez et al., 1991) and aminoacidemia (tudor et al., 1976) have been reported to have specific abnormalities in plasma amino acid profiles. there are some studies which make use of plasma amino acid profiles to diagnosis and distinguish abnormal subjects from healthy subjects, or subtypes and stages of diseases (noguchi et al., 2006) . one of the traditional examples of using plasma amino acid profiles for diagnostic markers is the fisher's ratio, which is a ratio of branched-chain amino acids to aromatic amino acids and is used for the marker of liver fibrosis (ferenci and wewalka, 1978; soeters and fischer, 1976) . these studies clearly show that plasma amino acid profile itself can be a useful tool for monitoring the physiological state of an organism. although these previous studies discuss how to distinguish different physiological states using the plasma amino acid profile data, the control mechanism behind the change in the profile is not thoroughly discussed. further investigations on the control mechanism of plasma amino acids should uncover the trigger reasons for diseases or propose a new treatment to improve the physiological conditions, however, the control mechanism is so complicated that the investigations have not been succeeded. the reasons for this complexity should come from the interrelatedness of the amino acids and a number of internal and external factors affecting their concentrations. amino acids are directly and indirectly related to each other within a large metabolic pathway and their concentrations in the plasma are equivalent to the whole sum of the metabolic flow in each organs and tissues. the metabolic flow in organs and tissues are affected by various factors such as nutrition, diseases, exercise, and body composition, and so does the plasma amino acid profile. also, there are still unknown metabolic pathways, which are being discovered not only experimentally but also with using automated methods (chou et al., 2006) . from these reasons, the changes in the concentrations of plasma amino acids can not be simply explained by the topological network structure of metabolic pathway map. in the previous study, we have introduced the correlation based network analysis of plasma and tissue amino acids (noguchi et al., 2006) and demonstrated the similarity of the behavior of two amino acids in plasma and tissues. in this study, we carried out the network analysis one step further and introduced the directional relationship between plasma amino acids. for this purpose, we used plasma amino acid profile data of rats fed on a single amino acid-deficient (minus-one) diet. in this experimental model, the plasma concentration of deficient amino acid decreases drastically, which also affects the concentration of other amino acids. this resembles the changes observed in the gene expression profile of single gene knock-out models (yeang et al., 2005) . taking advantage of this resemblance, the methods used for inferring gene expression network structure are adopted in this study for inferring the interrelated and directional network structure of plasma amino acids without considering any prior topological information on metabolic pathways. this data driven analysis should lead us to the further understanding of the dynamics of the plasma amino acid profiles and gives us some clues for the factors affecting the interrelated network under various physiological conditions. adult male sprague-dawley rats were maintained on a 12:12-h light-dark cycle, with water provided freely. starting from 10 weeks of age, the rats were randomly assigned and switched to control or one of the experimental diets (n ¼ 6 each). the composition of the diet is based on ain93g standard diet, slightly modified by replacing dextrin and sucrose by corn starch, and casein by amino acid mixture shown in table 1 . each experimental diet (minus-one diet) is completely depleted of single essential amino acids; histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. the rats fed branched-chain amino acid (bcaa) minus-one diets were kept for 5 weeks, and those fed other essential amino acid (eaa) minus-one diets were kept for 15 weeks under ad libitum feeding condition. rats were sacrificed 3 h after feeding and blood samples were collected from postcaval vein and mixed with edta immediately to prevent coagulation. the plasma separated from the blood samples are mixed with 2 volumes of 5% (w=w) trichloroacetic acid, and centrifuged immediately at 4 c, 8000 â g for 20 minutes. the supernatant was filtrated with ultrafree-mc filter (cat. no. ufc3lgc00, millipore), and the amino acid concentrations were measured by an automatic amino acid analyzer (l-8800; hitachi, tokyo, japan). in this study, we are to use threshold-test analysis and the multi-level digraph analysis methods to infer amino acid networks. using graphic approach or diagraph method to study biology-related can make the problem more intuitive, facilitating illustration and stimulating imagination so as to help reveal the essence of the problem concerned. graphic approach has been successfully used to study enzyme-catalyzed system (chou, 1981 (chou, , 1989 chou and liu, 1981; kuzmic et al., 1992; lin and neet, 1990) , protein folding kinetics (chou, 1990) , hiv reverse transcriptase inhibition mechanisms (althaus et al., 1993a, b, c; chou et al., 1994) , and analysis of base frequencies in the anti-sense strands of human protein coding sequences (zhang and chou, 1996) . recently, the images of cellular automata were used to investigate hbv virus gene missense mutation (xiao et al., 2005a) , hbv viral infections (xiao et al., 2006b) , represent biological sequences (xiao et al., 2005b) , predict protein subcellular location (xiao et al., 2006a) , and analyze the fingerprint of sars coronavirus (wang et al., 2005) . our strategy for the inference of interrelated amino acid networks can be summarized as follow: (1) given data of fold-change in concentration of deficiency or over-consumption in one essential amino acid under the stationary state, the threshold-test analysis method is applied to infer binary relationships between target amino acids. (2) the multi-level digraph analysis method infers consistent minimal binary relationships starting from many binary relationships derived from the threshold-test analysis method. a threshold-test analysis method treats the data representing the binary relations of change in the concentration of amino acids. these relations describe the effects of one amino acid on the concentration of the other amino acids and are mainly provided by the changes in the state of amino acid concentrations (fig. 1) . upon setting an arbitrary threshold value of concentration ratio, one can extract binary relations between two amino acids directly by estimation of the relative change in concentration ratios (y f ) or by the statistical probability of change in average concentrations (y p ) from deficiency or over-consumption in one essential amino acid experimental data. the following method is explained using the concen-tration ratios (y f ) as an example. a set of amino acids is defined as s ¼ fa; b; c; . . .g. we assume here experiments are those of deficiency or over-consumption of one essential amino acid, and that the measurements of concentrations of many amino acids are performed simultaneously. the change in concentration of the amino acids resulting from the deficiency or over-consumption of one target amino acid relative to its concentration under normal conditions (control of amino acids) is examined and recorded. this change may be recorded as an increase, decrease, or no change. an concentration ratio matrix, e, is created from a set of deficiency in one amino acid (or over-consumption) experiments, in which each matrix element represents the real-valued ratio of amino acid concentration. for instance, the value of matrix element eða; bþ indicates the relative change (the concentration ratio) in concentration of amino acid 'b' in comparison to its concentration under control conditions caused by the deficiency (or over-consumption) in amino acid 'a'. thus the matrix e is defined as e ¼ fða; bþ; . . .g. the inference procedures of this network model are as follows: (0) obtain the concentration ratio matrix e using several sets of the amino acid concentrations resulting from deficiency or over-consumption of one essential amino acid. (1) using the concentration ratio matrix e, we determine whether a given amino acid affects another given amino acid. for example, if, following the deficiency of amino acid 'a', the ratio of amino acid 'b' becomes higher than a given threshold value (specifically, more than y f -times higher), or becomes lower than a given threshold value (specifically, less than 1=y f -times lower) we say that amino acid 'a' affects amino acid 'b' directly or indirectly, and the value of element (a, b) in the binary matrix r is set to 1; r(a, b) ¼ 1. (2) it should be noted that the condition amino acid 'a' affects amino acid 'b', that is r(a, b) takes the value 1, means both ''a change in concentration-value of 'a' leads to a change in that of 'b''' and also ''no change in the concentration-value of 'b' leads to no change in that of 'a'''. thus the probability of rða; bþ takes the value 1, or more generally the probability that the value rði; jþ takes the value 1, pðrði; jþ ¼ 1þ (where i; j ¼ 1; 2; . . . ; n in which n is the total number of target amino acid) is examined through all the experiments of deficiency or over-consumption of one essential amino acid. an arbitrary second threshold value for probability is set, r, and experimental events with pðrði; jþ ¼ 1þ > r are extracted statistically. a multi-level digraph analysis method infers amino acid networks by using a set of binary relations between amino acid (e.g. amino acid 'a' affects (maki et al., 2001 (maki et al., , 2004 . a systematical analysis of the binary relations between pairs of amino acids enables us to reconstruct a possible minimum architecture of the amino acid network that is consistent with all of the data. in fig. 2 , the accessibility matrix r ã is derived directly from the binary relation r. in the accessibility matrix r ã , if there exists the relation that amino acid 'a' and 'b' affect each other, that is that r ã (a, b) ¼ r ã (b, a) ¼ 1, we cannot decide which amino acid is located at the upper stream. we therefore introduce an ''equivalence set'', which makes a single set of the group of amino acids affect each other, and this group is deemed to be a single amino acid. in order to partition amino acids into equivalence sets, we use the accessibility matrix r ã . this matrix is a relative transitive closure of the binary relation matrix, r, where the matrix entry r ã ða; bþ indicates whether amino acid 'a' finally affects amino acid 'b' or not. the multi-level digraph analysis model is implemented on the basis of this accessibility matrix r ã . figure 2 shows the procedure for drawing a multi-level digraph. for the accessibility matrix r ã in the figure, since 'c' and 'd' can be regarded as an equivalence set, we can combine them as 'c ã '. in this manner, we can draw up equivalence sets in a semi-ordered (topologically sorted) accessibility matrix. a semi-ordered accessibility matrix between equivalence sets includes indirect amino acid relations. in order to remove them and to make a skeleton matrix, we process the semi-ordered matrix as follows: the value of line i and column j in a semi-ordered matrix a and skeleton matrix s are represented as aði; jþ and sði; jþ, respectively. if aði; jþ ¼ 1, sði; kþðk ¼ 1; . . . ; nþ is set to maxfaði; jþ à að j; kþ; 0g. thus, all indirect effects are removed from the semi-ordered matrix. in fig. 2 , the relation between amino acid 'a' and 'c ã ' are removed, we thus can construct the skeleton matrix s. finally we draw lines between nodes based on the value fig. 2 . process of the multi-level digraph analysis method fig. 3 . plasma amino acid concentration ratio. the concentration of plasma amino acid by amino acid minus-one diet is shown as the ratio to that of control diet of each element in the skeleton matrix. in fig. 2 , the amino acids with parentheses indicate an equivalence set of amino acids. the sample plasma was obtained from the amino acid minus-one diet fed rats, whose plasma concentration of the deficient amino acid is less than half of that of the control diet fed rats. deficiency in one essential amino acid triggers the change in concentrations of all the other amino acids in plasma as shown in fig. 3 . this change is converted to the directional relation from the deficient amino acids to all the other amino acids by the described method. two different values were adopted to set the threshold. one is the fold-change value (y f -value). larger y f -value means the severer filtering condition. the other value is the p-value (level of significance) for the average difference (y p -value). each experimental and control groups consist of 6 samples, and for all the amino acids measured, the p-value, level of significance, for the average difference between the experimental group and the control group was calculated using dunnet's multiple comparison method. in this case, smaller y p -value means the severer filtering condition. network structure estimated by the multi-level digraph method from the binary interaction matrix, multi-scale digraph was drawn (fig. 4) . in the analysis using fold-change value as the threshold, the number of amino acids (nodes) composing the network decreases as the filtering threshold becomes severer. in the analysis using the p-value (level of significance), the number of nodes does not change drastically as the threshold changes. in either analysis, some amino acids formed an equivalence group, in which the interactions (links) form a loop and the direction of the effect on one another cannot be fig. 4 . network structure estimated by threshold-test analysis and multi-level digraph analysis methods. network structure of plasma amino acids is estimated using threshold-test analysis and multi-level digraph analysis methods. the amino acids whose minus-one diet experiments were performed are indicated in circles. the change in plasma amino acid concentration was converted to binary relational values, in the manner described in the results. the threshold to determine the binary value is set using a fold-change (y f -value) and b p-value (y p -value). for both cases, the figures to the right uses severer threshold in determining the binary values. lines with black arrowheads indicate the positive effects and the lines with white arrowheads indicate the negative effects decided. when the filtering condition becomes severer, the link forming the loop is disappeared and the equivalence group disperses into the smaller groups or individual amino acids. in the case using fold-change value, the equivalence group disperses at the threshold of 3.0, and 3-level digraph with 9 amino acids is drawn. in the case using p-value, the equivalence group does not completely disappear and at the threshold p ¼ 0.001, which is the severest condition inspected, 4-level digraph with 11 amino acids is drawn. since we should like to infer network model with as many nodes and links as possible, we decided to use p-value for the threshold for the further analysis. the same analysis was carried out using the partial dataset. by excluding one amino acid minus-one dataset, we can estimate the network structure without considering the effect of the excluded amino acid. the results are shown in fig. 5 . at the threshold p ¼ 0.05, the removal of threonine minus-one dataset changed the network structure drastically. the equivalence group disappears and the interactions between the amino acids which belonged to the group are alternatively revealed. this indicates that the relation responsible for holding the equivalence group together was the effect of threonine on the other amino acids, leucine, methionine, tryptophan and valine, in the group. at the threshold p ¼ 0.001, the only amino acids forming the equivalence group are threonine and methionine. by excluding threonine minus-one dataset, the effects of methionine on other amino acids are revealed, and by excluding methionine minus-one dataset, the effects of threonine are revealed. integrating the information obtained from the analysis of partial dataset, the estimated network structure is further refined to the model shown in fig. 6 . threonine directly interacts with the most number of amino acids, and lysine is located at the top control level in all the other amino acids. threonine and methionine is interrelated each other, which forms a feedback loop. using the data obtained under the essential amino acid minus-one condition, we have first estimated the coarse network structure of the plasma amino acids. the minus-one condition lowers the plasma concentration of the deficient amino acid to less than half, and this condition was maintained for a fairly long period. this drop in the concentration and the consequent changes in other amino acid concentrations are theoretically equivalent to the single gene disruption experiments in which the expression of the disrupted gene drops to nearly zero and the expression of the other genes are altered. in these experiments the genes whose expression levels were altered are suggested to be directly or indirectly regulated by the disrupted genes. similarly, the amino acids whose plasma concentration changed under minus-one condition are suggested to be directly or indirectly regulated by the deficient amino acid. combining the essential amino acid minus-one datasets, we were able to draw a coarse network model which can explain the change in the concentration of the plasma amino acids in our experiments. in the estimated plasma amino acid network structure, lysine is located at the top control level, which affects most of the amino acids, but is not influenced by any amino acids. this indicates the biological essentiality of lysine in an organism. lysine deficiency is the strongest signal and the wide range of amino acid metabolism has to be modulated to mitigate the damage. threonine interacts directly with the most number of the amino acids, acting as a hub in the network. this suggests the possible role of plasma threonine as a messenger which spreads the information of any essential amino acid deficiency to the whole body. when rats are fed minus-one diet, they will respond by changing their metabolic flow, to save and recycle the limited amino acid (kimball, 2002) and compensate for the shortage by making use of internal amino acid pool, such as skeletal muscle (kadowaki and kanazawa, 2003) . in this regulation of metabolic rate, threonine might play an important role. the concentration of threonine rises in any amino acid minus-one condition, except for threonine minus-one. thus it is highly possible that threonine may be used to regulate the pathway which will be needed in any essential amino acid shortages. as shown in fig. 6 , we can speculate that threonine gives positive influence to methionine, and methionine gives negative influence to threonine. these positive and negative interactions make up a loop which causes the temporal oscillatory behavior of the threonine-methionine concentration. this kind of oscillation can play a role in a trigger switch in a biological system. one of the mutual relationships that link threonine and methionine is the common catabolic pathway downstream of 2-oxobutanoate. threonine is deaminated to form 2-oxobutanoate (kapke and davis, 1976; scarselli et al., 2003) , and methionine forms 2-oxobutanoate via l-homocysteine and cystathionine . this can partly explain the positive effect of threonine on methionine. when the concentration of plasma threonine decreases, the concentrations of 2-oxobutanoate may also decrease and the methionine catabolism may be stimulated. cystathionine beta-synthase usually catalyzes the reaction of replacing beta-oh of serine by homocysteine, however, it is reported that threonine can be substituted for serine in this reaction forming 3-methylcystathionine (borcsok and abeles, 1982) . cystathionine beta-synthase is a key enzyme in methionine metabolism which directs the metabolite flux towards catabolic trans-sulfuration pathway rather than methionine recycling salvage pathway (banerjee and zou, 2005) . the fact that threonine can be a counter substrate for this enzyme indicates the possibility of threonine having direct regulatory effect on methionine metabolism. another possible link between threonine and methionine is vitamin b12. there are two enzymes which require vitamin b12, l-methylmalonyl-coa mutase and methionine synthase. l-methylmalonyl-coa mutase catalyzes the conversion of l-methylmalonyl-coa to succinyl-coa. l-methylmalonyl-coa is one of the metabolites of threonine. methionine synthase catalyzes the conversion of 5-ch 3 -tetrahydrofolate and homocysteine to tetrahydrofolate and methionine, respectively watanabe and nakano, 1999) . this may be a clue which explains the negative effect of methionine to threonine. the model reported in this study is constructed using the static plasma amino acid data obtained in a condition where plasma amino acids concentration had been equilibrated by the minus-one condition. it is a snapshot or a cross section, taken under this particular minus-one condition, of the dynamic plasma amino acid network. it is constructed without any prior topological information of the amino acid metabolic pathway, however, it is notable that some relations in the model, such as the direct relation of phenylalanine to tyrosine, can be explained by the pathway map. by comparing and integrating snapshots taken under various conditions, we can refine and validate the estimated network structure of the plasma amino acid. metabolic abnormalities in cobalamin (vitamin b12) and folate deficiency steady-state kinetic studies with the non-nucleoside hiv-1 reverse transcriptase inhibitor u-87201e kinetic studies with the non-nucleoside hiv-1 reverse transcriptase inhibitor u-88204e the quinoline u-78036 is a potent inhibitor of hiv-1 reverse transcriptase redox regulation and reaction mechanism of human cystathionine-beta-synthase: a plp-dependent hemesensor protein mechanism of action of cystathionine synthase two new schematic rules for rate laws of enzymecatalysed reactions graphic rules in steady and non-steady state enzyme kinetics applications of graph theory to enzyme kinetics and protein folding kinetics. steady and non-steady-state systems graphical rules for non-steady state enzyme kinetics kinetics of processive nucleic acid polymerases and nucleases predicting networking couples for metabolic pathways of amino acid metabolism in 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analysis of biological nmr spectroscopic data network analysis of plasma and tissue amino acids and the generation of an amino index for potential diagnostic use structure and function correlations between the rat liver threonine deaminase and aminotransferases insulin, glucagon, aminoacid imbalance, and hepatic encephalopathy elevation of serum cystathionine levels in patients with cobalamin and folate deficiency a study of aminoacidemia and aminoaciduria in epileptic children a new nucleotide-composition based fingerprint of sars-cov with visualization analysis serum amino acid levels in patients with hepatocellular carcinoma serum neutral amino acid concentrations in cirrhotic patients with impaired carbohydrate metabolism an application of gene comparative image for predicting the effect on replication ratio by hbv virus gene missense mutation using cellular automata to generate image representation for biological sequences using cellular automata images and pseudo amino acid composition to predict protein subcellular location a probability cellular automaton model for hepatitis b viral infections validation and refinement of gene-regulatory pathways on a network of physical interactions an analysis of base frequencies in the antisense strands corresponding to the 180 human protein coding sequences authors' address: nahoko shikata, graduate school of systems life sciences, kyushu university, 6-10-1 hakozaki, higashi-ku, fukuoka 812-8581, japan, fax: þ81-44-244-9617, e-mail: shikata@brs.kyushu-u.ac.jp key: cord-335316-x2t5h5gu authors: madariaga, m. l. l.; guthmiller, j.; schrantz, s.; jansen, m.; christenson, c.; kumar, m.; prochaska, m.; wool, g.; durkin, a.; oh, w. h.; trockman, l.; vigneswaran, j.; keskey, r.; shaw, d. g.; dugan, h.; zheng, n.; cobb, m.; utset, h.; wang, j.; stovicek, o.; bethel, c.; matushek, s.; giurcanu, m.; beavis, k.; disabato, d.; meltzer, d.; ferguson, m.; kress, j. p.; shanmugarajah, k.; matthews, j.; fung, j.; wilson, p.; alverdy, j. c.; donington, j. title: clinical predictors of donor antibody titer and correlation with recipient antibody response in a covid-19 convalescent plasma clinical trial date: 2020-06-23 journal: nan doi: 10.1101/2020.06.21.20132944 sha: doc_id: 335316 cord_uid: x2t5h5gu background: convalescent plasma therapy for covid-19 relies on the transfer of anti-viral antibody from donors to recipients via plasma transfusion. the relationship between clinical characteristics and antibody response to covid-19 is not well defined. we investigated predictors of convalescent antibody production and quantified recipient antibody response in a convalescent plasma therapy clinical trial. methods: multivariable analysis of clinical and serological parameters in 103 confirmed covid-19 convalescent plasma donors 28 days or more following symptom resolution was performed. mixed effects regression models with piecewise linear trends were used to characterize serial antibody responses in 10 convalescent plasma recipients with severe covid-19. results: mean symptom duration of plasma donors was 11.9 and 7.8% (8/103) had been hospitalized. antibody titers ranged from 0 to 1:3,892 (anti-receptor binding domain (rbd)) and 0 to 1:3,289 (anti-spike). multivariable analysis demonstrated that higher anti-rbd and anti-spike titer were associated with increased age, hospitalization for covid-19, fever, and absence of myalgia (all p<0.05). fatigue was significantly associated with anti-rbd (p=0.03) but not anti-spike antibody titer (p=0.11). in pairwise comparison among abo blood types, ab donors had higher anti-rbd titer than o negative donors (p=0.048) and higher anti-spike titer than o negative (p=0.015) or o positive (p=0.037) donors. eight of the ten recipients were discharged, one remains on ecmo and one died on ecmo. no toxicity was associated with plasma transfusion. after excluding two ecmo patients and adjusting for donor antibody titer, recipient anti-rbd antibody titer increased on average 31% per day during the first three days post-transfusion (p=0.01) and anti-spike antibody titer by 40.3% (p=0.02). conclusion: advanced age, fever, absence of myalgia, fatigue, blood type and hospitalization were associated with higher convalescent antibody titer to covid-19. despite variability in donor titer, 80% of convalescent plasma recipients showed significant increase in antibody levels post-transfusion. a more complete understanding of the dose-response effect of plasma transfusion among covid-19 patients is needed to determine the clinical efficacy of this therapy. convalescent plasma therapy has historically been used as a treatment during epidemics (1) . in this therapy, neutralizing anti-viral antibodies, as well as non-neutralizing antibodies and other immunomodulators, are transferred via plasma transfusion from those who have recovered from disease to those currently infected (2) (3) (4) . for patients with severe covid-19, convalescent plasma therapy has safely led to improvement in clinical and radiographic parameters (5) (6) (7) (8) (9) (10) . once adequate numbers of people convalesced and supply chain logistics were established, providing plasma therapy to a large number of patients has proven feasible (11) . efficacy of convalescent plasma therapy relies on a robust antibody response in convalescent plasma donors. measurements of antibody response among patients with covid-19 demonstrate that the majority develop igm and igg within 2 weeks of symptom onset, with specificity towards receptor binding domain (rbd) and spike protein viral epitopes correlating with virus neutralization (12) (13) (14) . strikingly, a small proportion of recovered covid-19 patients show no detectable antibodies to these epitopes (12, 15) . the relationship between host characteristics, disease course and variability in antibody response to covid-19 is poorly understood. the aim of this study was to establish a translational convalescent plasma program to investigate the relationship between clinical and serological parameters in convalescent plasma donors and define the antibody response of convalescent plasma recipients. this was a prospective open label clinical study to assess the feasibility, safety and immunological impact of delivering anti-sars-cov-2 convalescent plasma to hospitalized patients aged 18 years or older with severe or life-threatening covid-19 disease within 21 days from the onset of their illness. this study was conducted at university of chicago medicine (ucm) from april 10, 2020 to may 17, 2020 . the final date of follow-up was may 25, 2020. we used existing hospital infrastructure and personnel to build the convalescent plasma program at a time when state-wide shelter-in-place orders were active, elective procedures were not being performed, and non-covid-19-related research activities were halted. the donor enrollment team consisted of two surgeons, two surgical residents, and three physician assistants. a dedicated study coordinator was present at the ucm blood donation center to facilitate whole blood donation and collect research samples. recipients were selected during daily videoconference with infectious disease. one surgeon visited the hospital covid-19 unit daily to obtain consent and research samples. plasma donors were age 18 or older, able to donate blood per standard ucm blood donation center guidelines, had a documented covid-19 polymerase chain reaction (pcr) positive test, and complete resolution of symptoms at least 28 days prior to donation. recruitment occurred via social media, news outlets, word-of-mouth and announcements in university and community bulletins. the ucm infectious disease team provided an institutional list of patients with a positive pcr test for covid-19, and their physicians were emailed to request permission to contact the patient for donor participation. interested plasma donors were directed to fill out a short screening survey online. potential donors meeting study criteria were screened for eligibility, reported symptoms and comorbidities, consented, and were scheduled for donation at the ucm blood donation center in a single telephone encounter. after meeting the ucm blood donation center eligibility, whole blood was collected and processed according to standard ucm blood donation center procedures. standard whole blood donation was used for plasma collection because it fit into preexisting ucm blood bank infrastructure and workflow therefore facilitating rapid deployment of a collection process, and allowed red blood cell and unused plasma units to be used in the regular blood bank inventory. during blood donation, a single research sample was collected at the same time as blood samples for standard immunohematology testing and infectious disease screening. leukocyte filters used in separation of constituent blood parts were also collected for research. eligibility for convalescent plasma recipients included: age 18 or older, laboratoryconfirmed covid-19, within 21 days from the start of illness and severe or life-threatening covid-19 as defined by the united states food and drug administration (fda) (16). severe covid-19 was defined as dyspnea, respiratory frequency ≥ 30/min, blood oxygen saturation ≤ 93%, partial pressure of arterial oxygen to fraction of inspired oxygen ratio < 300, and/or lung infiltrates > 50% within 24 to 48 hours. life-threatening covid-19 was defined as respiratory failure, septic shock, and/or multiple organ dysfunction or failure. patients who were pregnant, received pooled immunoglobulin in the past 30 days or had a history of transfusion reaction were excluded from this study. recipients had routine pre-transfusion testing, in keeping with institution policies. on the day of enrollment, an emergency investigational new drug (eind) application was filed and approved for each recipient by the fda (16). subsequently, one abo-compatible unit of convalescent plasma (~300 ml) was transfused over 4 hours. repeat administration of convalescent plasma occurred in one recipient (r7). blood samples and nasopharyngeal swabs were obtained at day 0, 1, 3, 7, 14 post transfusion. the primary outcome was feasibility as defined by the collection of convalescent plasma and its administration into hospitalized patients. secondary outcomes included type and duration of respiratory support, cardiac arrest, transfer to intensive care unit (icu), length of stay, mortality, complications of plasma administration, process outcomes, and antibody titer of plasma donors and recipients. levels of anti-rbd and anti-spike antibodies were measured by enzyme-linked immunosorbent assay (elisa) in blood samples at time of donation and plasma recipients, as previously described (17) . nasopharyngeal specimens were obtained by flocked swabs in plasma recipients and analyzed by rt-pcr to detect sars-cov-2 rna. study data were collected and managed using redcap electronic data capture tools hosted at ucm (18, 19) . donor patient characteristics were compared using the chi-squared test for categorical variables and the two-sample t test for continuous variables. univariate regression analysis for antibody titer (anti-rbd and anti-spike) was conducted against age, sex, body mass index (bmi), previous pregnancy, previous blood donation, blood type, symptoms (fever, cough, sore throat, dyspnea, abdominal pain, aguesia, anosmia, fatigue, myalgia, headache), co-morbidities (respiratory, cardiovascular, renal, diabetes, autoimmune disease, cancer, liver disease), smoking history, travel in the past 3 months to the united states, asia or europe, symptom duration, interval from symptoms resolution to plasma donation, and hospitalization. pairwise comparison using t tests without adjusting for multiple comparisons was used to compare antibody titers among different abo blood groups. we conducted multivariable analyses to identify prediction models for anti-rbd and anti-spike antibody titers among convalescent plasma donors. best subset variable selection method was chosen to identify the subset of predictors that maximizes the adjusted r-squared among all possible models. to compare daily change in recipient antibody response, we fit mixed effects regression models with piecewise linear trend with a change point at 3 days after intervention for log-transformed antibody titers. we considered recipients on extra-corporeal membrane oxygenation (ecmo) (r3 and r6) separately from recipients not on ecmo (r1, 2, 4, 5, 7, 8, 9, 10), because ecmo recipients had different baseline characteristics. data analysis was performed using software r, version 3.6.3. mixed effects regression models were fit using the lmer function of the lme4 package (20) . data analysis was conducted within rstudio environment, and r markdown files with fully reproducible data analysis can be obtained from the authors upon request. this study was approved by the institutional review board (irb20-0523). all participants (plasma donors and plasma recipients) gave written informed consent prior to inclusion in the study. analysis was performed by mlm and mg. this clinical trial was registered at clinicaltrials.gov with identifier nct04340050. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. . https://doi.org/10.1101/2020.06.21.20132944 doi: medrxiv preprint 697 potential plasma donors were recruited to our study over 35 days (table 1 ). the average age was 43.5 years (range 18 to 87), the majority were female (63.1%), and 37% had never donated blood before. potential donors with confirmed positive covid-19 pcr (n=384, 55%) were more likely to be male, have ageusia and anosmia, and lack cough, sore throat and dyspnea compared to the 313 symptomatic patients who had clinical signs of covid-19 but were never tested (table 1) . among plasma donors (n=103) who donated as of publication, average symptom duration was 11.9±5.91 days, 9 (8.7%) had respiratory comorbidities such as asthma, chronic obstructive pulmonary disease or obstructive sleep apnea, and 8 (7.8%) had been previously hospitalized for covid-19 ( table 1 ). the average interval between symptom start and plasma donation was 45.1±8.02 days. donor antibody titers measured on day of plasma donation ranged from 0 to 1:3,892 (anti-rbd) and from 0 to 1:3,288.7 (anti-spike) ( table 1 ). in univariable regression analysis, higher average anti-rbd and anti-spike antibody titers were associated with plasma donors who were older, male, had higher bmi, had fever and had been hospitalized (p<0.05, supplemental table 1 ). in a pairwise comparison among abo groups without adjusting for multiple comparisons, ab donors had higher anti-rbd titer than o negative donors (p=0.048) and higher anti-spike titer than o negative (p=0.015) or o positive (p=0.037) donors. to determine predictors of anti-rbd and anti-spike antibody titer, we performed best subset multivariable analysis including age, sex, blood type, history of previous blood donation, fever, cough, fatigue, myalgia, symptom duration, hospitalization and travel in the united states within the past 3 months. significant predictors of anti-rbd antibody titer were age (p=0.02), fever (p<0.01), previous hospitalization (p<0.01), lack of myalgia (p=0.01), and fatigue (p=0.03) (r-squared=0.40, adjusted r-squared=0.32, table 2 ). significant predictors of anti-spike antibody titer were age (p=0.02), fever (p=0.01), previous hospitalization (p=0.01), and absence of myalgia (p<0.01) (r-squared=0.35, adjusted r-squared=0.26, table 2 ). o positive blood type was associated with lower anti-rbd (p=0.05) but did not meet significance threshold for antispike (p=0.07). ten hospitalized patients with severe or life-threatening covid-19 received plasma on day 0 ( figure 1 , table 3 ). plasma recipients were on average 61.9 years old (range 30 to 86) and 40% female. the average time from start of symptoms to plasma transfusion was 12 days (range 2 to 21) and the average time from hospital admission to plasma transfusion was 6 days (range 2 to 17). at the time of plasma transfusion, two patients were on ecmo, one patient was mechanically ventilated, two patients were on high-flow nasal cannula (hfnc), four patients were on nasal cannula and one patient was on room air. five patients had received other therapies for covid-19 before transfusion, including remdesivir, tocilizumab, anakinra and hydroxychloroquine. two plasma recipients were on chronic immunosuppression after transplantation. figure 2 shows selected clinical and laboratory parameters of convalescent plasma recipients. only one recipient (r8) had fever prior to transfusion and this resolved by day 3 post-transfusion. r3 and r6 remained on ecmo throughout the study period. in the remaining 8 recipients, oxygen requirements improved to room air or nasal cannula. the sequential organ failure assessment (sofa) score (21) was calculated for recipients on mechanical ventilation or ecmo and showed a general trend towards improvement; notably both ecmo patients were weaned off vasopressor and intra-aortic balloon pump support by 7 days post-transfusion. levels of inflammatory marker c-reactive protein (crp) were variable. crp decreased in six recipients (r1, r2, r5, r6, r9, r10). sars-cov2 np swab pcr remained positive in 5 patients and turned negative in 4 patients; 1 patient (r6) had been positive for sars-cov2 17 days prior to plasma transfusion but was negative for sars-cov2 on day of transfusion ( figure 1 ). at last follow-up, 1 patient on ecmo remained in the hospital (r6), 1 patient on ecmo was transitioned to comfort care and died on day 30 after plasma transfusion (r3), 4 patients were discharged to rehabilitation facilities and 4 patients were discharged to their place of residence ( figure 1 ). on day of transfusion, anti-rbd antibody titers were undetectable in 3 recipients (r1, r2, r10) and anti-spike antibody titers were undetectable in 3 recipients (r1, r8, r10) ( table 3 and figure 3 ). both patients on ecmo had very high antibody titer at day 0 which decreased in the days after transfusion (figure 3 ). the remaining plasma recipients showed increase in antibody titer within the first three days after transfusion (r1, 2, 4, 5, 7, 8, 9) with the exception of r10 who did not show any antibody titer until day 7 (anti-spike) and day 14 (anti-rbd) after transfusion ( figure 3 ). we performed a mixed effects model for log-transformed reciprocal antibody titer adjusting for donor antibody titer level looking at the first 3 days post-transfusion among the non-ecmo patients. after plasma transfusion, recipient anti-rbd antibody titer increased on average by 31% per day (p=0.01) and recipient anti-spike antibody titer increased on average by 40.3% per day (p=0.01) (figure 4 ). among the two ecmo recipients, recipient antibody response was not significantly changed until three days after plasma transfusion (decreasing by 9.2% per day for anti-rbd titer and 8.2% per day for anti-spike titer, p<0.01) (figure 4) . we monitored the clinical status of the recipients before, during and immediately after transfusion. no recipients experienced toxicity associated with plasma transfusion. there was no clinical deterioration or worsening of disease status immediately related to plasma transfusion. convalescent plasma transfusion was safe in high-risk individuals in our study: immunosuppressed patients after stem cell and lung transplants and a patient with end-stage renal disease on dialysis. we developed a translational convalescent plasma treatment program within the existing hospital infrastructure during the covid-19 pandemic that provided a new therapeutic option for patients while assessing the antibody profile of both convalescent and hospitalized patient populations. our multivariable analysis demonstrated that clinical characteristics can predict serological response of antibodies associated with virus neutralization (12) . higher anti-rbd and anti-spike antibody were more likely found in convalescents who were older, hospitalized, had fever, and lacked myalgia. fatigue also significantly predicted higher anti-rbd but not antispike antibody titer. variability in convalescent populations and immune response to viral infection may explain why recovery is not always marked by seroconversion (12, 15) . indeed, in our study four plasma donors (as well as four plasma recipients) had undetectable antibody titers. disparate plasma donor populations and geography may explain why symptom duration and elapsed time from symptom onset was associated with antibody response in new york city (13) but not among our patients in chicago. disparate plasma donor populations and geography may also explain antibody variability. these data highlights that the impact of variability in antibody type and titer on virus-neutralizing activity and long-term immunity is unknown. interestingly, we found that antibody titers significantly differed across abo blood type groups, (24) . further studies on the relationship between abo polymorphism and antibody titer may uncover genetic determinants of the host response to covid-19. recipients received plasma with a range of antibody titer from 1:73 to 1:3,892 (anti-rbd) and 1:69 to 1:2,921 (anti-spike). despite this, 80% of recipients demonstrated a significant increase in anti-spike and anti-rbd antibody titer in the 3 days post transfusion that was independent of donor antibody titer and were discharged after clinical improvement. interestingly, recipient antibody titer continued to increase up to 14 days in four recipients (r1, 2, 8, 10); in contrast, the two most severely ill patients on ecmo who had the highest antibody titers (up to 1:13,833 anti-spike antibody in r6) showed a decrease in antibody titer after receiving plasma on day 20-21 of illness. importantly we demonstrate the safety of transfusing convalescent plasma in immunosuppressed patients after lung transplantation and stem cell transplantation. none of the plasma recipients in this study deteriorated after convalescent plasma transfusion, consistent with the safety profile of other trials (5) (6) (7) (8) (9) 11) . repeat plasma dose in recipient r7 was also welltolerated. pre-clinical models of sars-cov and clinical experience of other viral illness had raised concern about the potential for non-neutralizing antibody to cause antibody dependent enhancement of disease, which was not seen here despite variable titers of donor antibodies (25) (26) (27) . the variability in post-transfusion recipient antibody titer and clinical response seen here and in other studies (5, 6, 28, 29) indicates that the therapeutic activity of convalescent plasma depends on the timing of treatment and composition of convalescent plasma. indeed, plasma contains more than 1,000 proteins, including albumin, immunoglobulins, complement, and coagulation factors as well as organic compounds such as cytokines (4) . convalescent plasma drawn shortly after natural infection (1, (5) (6) (7) (8) may be enriched for populations of protective antibodies not present in plasma derived from long-recovered or rarely-hospitalized donors studied here. furthermore, immunomodulatory and non-virus neutralizing antibody effects such as stimulation of the host humoral immune response and facilitating viral uptake into cells via fc-receptors to increase viral antigen presentation to other effector cells may contribute to disease recovery. taken together, while randomized controlled efficacy trials for convalescent plasma therapy in covid-19 are currently underway, establishing effective anti-covid-19 plasma-based therapy will require both an understanding of the precise dose and type of virusneutralizing antibody and in-depth characterization of plasma donor-recipient pairs. the availability of a pre-existing hospital-based blood collection facility within our medical center significantly eased the procurement of convalescent plasma and will allow us to assess immunological characteristics of donor-recipient pairs in future studies. such hospitalbased blood collection facilities have been declining in number across the united states for several decades (30) . cultivating region-specific convalescent plasma inventory may potentially facilitate the identification and isolation of antibodies with specific activity against local virus strains and be a useful model for future outbreaks. in addition, convalescent plasma derived from whole blood collection is a rapidly scalable technique that requires basic phlebotomy and blood separation rather than a dedicated apheresis personnel and equipment. furthermore, a significant proportion (36.3%) of our plasma donors had never donated blood before, indicating that a convalescent plasma donation program can serve as important community outreach during a time when patients avoid hospitals that are perceived as unsafe (31) . in summary, development of a convalescent plasma program is feasible, rapidly deployable and economical when existing resources of equipment, space and personnel are used. establishing the clinical predictors of high antibody titer and understanding the serological posttransfusion response may guide patient selection and shed light on antibody response to covid-19. further work characterizing convalescent plasma donor and recipient pairs is needed to elucidate mechanisms of convalescent plasma therapy and demonstrate optimal viral epitope therapeutic targets. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. . , interval of symptoms to plasma donation, blood type were not significantly associated with anti-rbd or anti-spike antibody titer. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. . https://doi.org/10.1101/2020.06.21.20132944 doi: medrxiv preprint treatment of influenza pneumonia by the use of convalescent human serum: preliminary report the convalescent sera option for containing covid-19 convalescent plasma as a potential therapy for covid-19 convalescent plasma in covid-19: possible mechanisms of action effectiveness of convalescent plasma therapy in severe covid-19 patients treatment of 5 critically ill patients with covid-19 with convalescent plasma treatment with convalescent plasma for critically ill patients with sars-cov-2 infection use of convalescent plasma therapy in two covid-19 patients with acute respiratory distress syndrome in korea patients with convalescent plasma in convalescent plasma treatment of severe covid-19: a matched control study early safety indicators of covid-19 convalescent plasma in 5,000 patients humoral immune response and prolonged pcr positivity in a cohort of 1343 sars-cov 2 patients in the new york city region temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup research electronic data capture (redcap)--a metadata-driven methodology and workflow process for providing translational research informatics support the redcap consortium: building an international community of software platform partners fitting linear mixed-effects models using lme4 the sofa (sepsisrelated organ failure assessment) score to describe organ dysfunction/failure. on behalf of the working group on sepsis-related problems of the european society of intensive care medicine abo blood group and susceptibility to severe acute respiratory syndrome relationship between the abo blood group and the covid-19 susceptibility inhibition of the interaction between the sars-cov spike protein and its cellular receptor by anti-histo-blood group antibodies anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection current studies of convalescent plasma therapy for covid-19 may underestimate risk of antibody-dependent enhancement treatment with convalescent plasma for influenza a (h5n1) infection use of convalescent plasma therapy in sars patients in hong kong continued decline in blood collection and transfusion in the united states-2015 delayed access or provision of care in italy resulting from fear of covid-19 we thank all the plasma donors for their willingness to help in a time of need and the blood bank staff for their excellent care. we thank samantha guerrero, alyssa anneken, bruce boehrnsen and rohit allada for helping us establish the infrastructure for this study. we thank the university of chicago and department of surgery, university of chicago for providing support for this study. this study was funded by the department of surgery, university of chicago and the national institute of allergy and infectious diseases (niaid) collaborative influenza aka, above the knee amputation; chf, congestive heart failure; dm, diabetes mellitus; dvt, deep venous thrombosis; esrd, endstage renal disease; htn, hypertension; nafld, non-alcoholic fatty liver disease; pe, pulmonary embolism; pvd, peripheral vascular disease.all rights reserved. no reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity.the copyright holder for this prep this version posted june 23, 2020. . https://doi.org/10.1101/2020.06.21.20132944 doi: medrxiv preprint key: cord-010328-uxpedpz8 authors: ciencewicki, jonathan m.; schouest, katherine r.; gierman, todd m.; vandeberg, peter j.; gooch, barry d. title: plasma donors in the southwestern united states positively contribute to the diverse therapeutic antibody profile of immune globulin products date: 2020-04-22 journal: sci rep doi: 10.1038/s41598-020-63794-y sha: doc_id: 10328 cord_uid: uxpedpz8 human-plasma-derived immune globulin (ig) is used in augmentation therapy to provide protective levels of antibodies to patients with primary immune deficiency diseases (pidd) and for prophylaxis against infectious diseases. to maintain the breadth of antibodies necessary for clinical protection, it is important to understand regional patterns of antibody seroprevalence in source plasma from which ig products are manufactured. in this study, source plasma from donation centers in various locations of the southwestern quarter of the united states was surveyed for antibody titers to hepatitis a virus (hav), measles virus (mev), and cytomegalovirus (cmv). a broad range of anti-hav ig plasma titers was observed among these centers, with some centers exhibiting 3–5 times the titers of the others. minor to no differences were observed for levels of anti-mev and anti-cmv, respectively. importantly, elevated anti-hav ig titers were broadly observed across plasma units obtained from the centers exhibiting high titers, indicative of a potential regional phenomenon among donors as opposed to few donors with singularly high titers. plasma from these high-titer centers conferred significantly greater neutralization against hav in vitro. the outcomes of this study give a glimpse of the antibody diversity inherent in human plasma used to manufacture ig products.. observed among these centers, with some centers exhibiting 3-5 times the titers of the others. minor to no differences were observed for levels of anti-mev and anti-cmv, respectively. importantly, elevated anti-hav ig titers were broadly observed across plasma units obtained from the centers exhibiting high titers, indicative of a potential regional phenomenon among donors as opposed to few donors with singularly high titers. plasma from these high-titer centers conferred significantly greater neutralization against hav in vitro. the outcomes of this study give a glimpse of the antibody diversity inherent in human plasma used to manufacture ig products.. immune globulin (ig) products made from human plasma are used in augmentation therapy to provide antibodies critical for the protection of persons with primary immune deficiency diseases (pidd), as well as for prophylaxis of various infectious diseases. the broadly-protective properties of the antibodies in ig products stem from their human origin and because they are purified from pools made up of large numbers of plasma donations. ig products manufactured in the united states (us), as well as some ig products manufactured in other countries, are derived from plasma collected exclusively from donation centers in the us, which can span the entire geography of the country. safety measures implemented nearly two decades ago-collaborative, industry-wide epidemiological surveillance of the donor population, judicious selection of donors, rigorous testing of plasma by serological and molecular (e.g., nucleic acid testing) methods and validation of manufacturing methods for virus clearance capacity-provide a high degree of confidence that ig products, regardless of plasma origin, safely deliver a diverse, natural combination of human polyclonal antibodies 1,2 . seroprevalence of antibodies to infectious diseases in the human population is influenced by a number of factors, including management of food and water resources, sanitation, vaccination, climate, sociodemographics, and the emergence of infectious agents, previously unknown or familiar 3 . since the status of factors such as these can vary over time and across geographic regions, the profiles of antibodies among donors whose plasma is used to manufacture ig products are dynamic. for example, changes in viral antigen exposure in recent years have had a real impact on ig products' antibody levels against a number of viruses, among which are hepatitis a virus (hav) and measles virus (mev). in the case of hav, declining overall rates of hepatitis a infection, due particularly to increased vaccine coverage beginning in the mid-1990s, have had the effect of reducing naturally-acquired anti-hav titers among adult plasma donors [4] [5] [6] [7] [8] [9] . consequently, available ig preparations have been found to fall short of potency expectations 10 . this development has prompted grifols, manufacturer of the lone ig product (gamastan ® ) approved in the us for hav prophylaxis, to update dosage regimens to ensure that adequate circulating anti-hav immunoglobulin levels are reached in patients 11, 12 . the use of widespread measles vaccination has had a similar effect on mev-neutralizing antibodies in ig products for intravenous administration 13 . nevertheless, human plasma ig augmentation therapy still offers the unique advantage of a diverse, polyvalent therapeutic antibody repertoire that cannot be obtained by any means other than purification of ig from large numbers of plasma donations collected across a wide geographic spectrum. a better understanding of the antibody diversity with respect to geography could help to mitigate the decline in antimicrobial potencies observed in ig preparations. for example, it is known that anti-hav seroprevalence in the us is greatest among populations residing in the southwestern us 14,15 , a phenomenon not necessarily due to expanded immunization coverage, but more likely indicative of more potent, longer-lived naturally-acquired immunity. the present study was designed primarily to examine the anti-hav content, and potential benefit, of plasma obtained from donors in the southwestern quarter of the us. in addition, since it is possible this plasma possesses elevated antibody levels to other infectious agents 16 , we examined levels of antibodies to mev and human herpesvirus 5, also known as cytomegalovirus (cmv). human plasma. plasma was obtained from donation collection centers located in el paso, texas (tx); mcallen, texas (tx); san diego, california (ca); midwest city, oklahoma (ok); and provo, utah (ut), all of which are associated with the southwestern us. plasma was also obtained from one center, clarksville, tennessee (tn), outside of this region. the primary selection criterion for donation centers was proximity to regions of high hav incidence and seroprevalence. while texas and california have some of the highest incidence and seroprevalence rates in the us, oklahoma and utah-which are loosely associated with the southwest-and tennessee have relatively low incidence rates. texas and california also have a higher foreign born population than the other states, which may contribute to the higher seroprevalence. beyond the selection of states, criteria were: (1) centers that perform testing at the facility to allow for quick access to samples; (2) centers that have sufficient unique donors over a short time-span to produce an adequate sample size. donors are allowed to donate two times per week, and since unique donors were desired, it was prudent to limit the collection period of samples to a few days in order to avoid duplicate donors. all plasma samples used in this study were residuals of samples processed as part of grifols' standard routine for the qualification of donors and material for further manufacture. each donor provides informed consent that allows grifols to conduct research on samples collected at its plasma collection centers. the informed consent documentation has been reviewed by institutional review boards (irbs) as part of several prospective clinical studies. all plasma units from which samples for this study were pulled were non-reactive for all serological and nucleic acid tests performed, including hav ribonucleic acid (rna) by real-time quantitative reverse transcription (qrt) polymerase chain reaction (pcr) testing. for the purpose of this study, to allow for higher throughput antibody testing, plasma pools were created by combining aliquots of 16 virus propagation. b-sc-1 cells (seeded 24 h prior) were infected with hav (hm175/18 f; atcc) at a multiplicity of infection (moi) of 0.05 tcid 50 per cell. after incubation at 35 °c for 7 d, a cell lysate was prepared and stored frozen at −70 °c. thawed lysate was treated with trixton-x 100 and lithium dodecyl sulfate and concentrated via tangential flow filtration (tff). the tff retentate was centrifuged at approximately 85000 × g for 1.5 h at 5 °c in order to force the virus into a pellet, after which the pellet was suspended in buffer and the buffer was exchanged by ultrafiltration. the virus preparation was stored at −70 °c until use. a strain of cmv (ad-169; atcc) frequently used in laboratories has been passaged extensively in mrc-5 fibroblasts such that it no longer exhibits tropism for naturally permissive epithelial/endothelial cells. in order to obtain a more clinically-relevant system, endothelial arpe-19 cells were infected with ad-169 at an moi of 0.1-1.0 tcid 50 per cell, and the cells were serially passaged post infection until a desirable cmv titer was achieved. adaptation of the virus did not impact its ability to infect mrc-5 fibroblasts (data not shown). the supernatant from infected cells was used without further purification in neutralization experiments. enzyme-linked immunosorbent assay. a commercially-available enzyme-linked immunosorbent assay (elisa) (bio-rad monolisa ™ anti-hav eia kit, cat#72496) was used to estimate the anti-hav ig (igg and igm) titer in a plasma sample. an in-house protocol was followed to convert the elisa to a semi-quantitative format. with each assay plate was included a series of dilutions of the kit's calibrator, from which a standard curve was generated. a plasma sample was diluted such that its absorbance value was within the range of the standard curve, and an ig titer was estimated from a linear fit of the standard curve. the ig titer was then corrected according to the dilution factor. commercially-available elisas (abcam anti-cmv igg human elisa kit cat#ab108724 or abcam anti-mev virus igg human elisa kit cat# ab108750) were used to semi-quantitatively determine the amount of anti-cmv or anti-mev igg in a plasma sample. the assay was performed according to the manufacturer's instructions. a single plasma sample, neat or diluted in hank's buffered salt solution (hbss), was added to the first well of each row of a 96-well microtiter plate. an equal volume of hbss was added to the remaining wells of the entire plate. the plasma in these first wells was serially diluted across the succeeding nine wells of a respective row. an equal volume of a virus preparation was added to the same wells such that the concentration of virus in each well was 6.0 to 6.5 tcid 50 ·ml −1 . a positive control was included on each plate by adding the virus preparation to the wells of a single column containing only hbss. a negative control was also included in which hbss alone was left in the wells of a single column. the plate was then incubated for 60 min at 37 °c. following the incubation, 50 µl from each well were used to inoculate a corresponding well on a microtiter plate that had been seeded with recipient cells (frhk for hav and arpe-19 for cmv; mev were not evaluated). the recipient cells were incubated with the inoculum for 2 h at 37 °c. following infection, the inoculum was removed, maintenance medium was added, and the cells were incubated at 35 °c for hav or 37 °c for cmv. twenty-one days post inoculation, the cells were microscopically examined for the visual appearance of a cytopathic effect (cpe). the virus neutralization factor of a given plasma sample was calculated as the reciprocal of the largest dilution at which not less than 50% of its inoculated wells were negative for cpe. in this case, each dilution had eight replicates (the eight wells comprising a column of a microtiter plate); therefore, the last dilution in the series that exhibited at least four cpe-negative wells dictated the neutralization factor for the plasma sample on test. statistical analyses. statistical analyses of data were performed using sas jmp (v14) and graphpad prism (v8) software. for analyses of grouped data sets, an unpaired two-tail t test was used to determine if means differed significantly. for the analysis of collection centers, a one-way analysis of variance (anova) was used to determine if the means differed significantly, and tukey's honestly significant difference (hsd) post hoc test was used to directly compare means of each center. anti-hav, -mev, and -cmv immune globulin seroprevalence in source plasma from us southwest. all 171 plasma pools were screened by an elisa for anti-hav ig (igg and igm). when analyzed per center (fig. 1) , the mean anti-hav ig titers of the plasma pools from el paso-tx (5,267 ± 1,300 miu·ml −1 ) and mcallen-tx (4,838 ± 1,406 miu·ml −1 ) were comparable to each other and significantly (p < 0.0001) higher than the mean titers of all other centers. the mean anti-hav ig titers of pools from the san diego-ca (2,296 ± 2,052 miu·ml −1 ) and midwest city-ok (1,665 ± 1,131 miu·ml −1 ) centers were comparable; the mean titer of san diego-ca pools was significantly (p < 0.001) higher than those of provo-ut and clarksville-tn. the 32 plasma pools from el paso-tx and the 25 pools from midwest city-ok were analyzed for anti-mev and anti-cmv igg content (fig. 2) . the level of anti-mev igg calculated for midwest city-ok pools was found to be slightly higher compared to that of pools from el paso-tx (p < 0.0001). there was no significant difference in levels of anti-cmv igg between the two sets of pools. titers were due to one or a few donors in a given pooled sample or due to an overall higher prevalence in the population, samples from the individual donations comprising twelve of the plasma pools were tested for anti-hav ig titers. it was necessary to choose a subset of the pools in order to maintain a feasible laboratory workload. three pools each from the el paso-tx, mcallen-tx, midwest city-ok, and clarksville-tn centers were arbitrarily selected from within the second and third quartiles of the anti-hav ig titers for their respective centers. for samples of individual donations that had composed the pools from el paso-tx and mcallen-tx, a broad range of anti-hav ig titers were observed, but less than 15% of the donations exhibited values above 10,000 miu·ml −1 . the mean and median titers of the donations making up each pool never differed by more than approximately 2.5-fold (fig. 3) . a much lower mean anti-hav ig titer and a narrower range of titers were observed for the samples of individual donations pooled from centers in the other locations. assessment of antiviral activity. the same twelve pools identified for analysis of their component donations, as described in the prior section, were also evaluated for their capacity to inhibit hav and cmv infection in vitro (fig. 4a,b) . neutralization of hav infectivity resulting from plasma pools obtained from the el paso-tx center (mean neutralization factor of 4625) was significantly (p < 0.001) higher than what was observed for plasma pools from the midwest city-ok center (mean neutralization factor of 1042). no significant difference was observed in the capacity to neutralize cmv. neutralization of mev was assayed and activity was detected, but the results were inconclusive. the method had been optimized for concentrated ig samples, not for raw plasma, and did not possess sufficient sensitivity to distinguish differences in titers between plasma samples. further evaluation was not deemed necessary. the use of pooled, plasma-derived human ig has become a critical therapy in clinical medicine [17] [18] [19] . while originally indicated as a plasma protein augmentation therapy for patients with pidd and some secondary immunodeficiency diseases, ig has also been shown to exhibit other clinical benefits, many stemming from its anti-inflammatory and immunomodulatory effects 20, 21 . it is the diverse, polyclonal nature of ig that has endowed it with its broad clinical range. in order to maintain the therapeutic diversity of ig products, it is critical to understand patterns of antibody seroprevalence in source plasma. to this end, we tested plasma obtained from donor centers within the southwestern quarter of the us. the data confirm that the notable anti-hav ig seroprevalence in particular areas of the us southwest translates into elevated anti-hav ig titers in plasma collected at donation centers in those areas. clearly, elevated antibody levels specific for hav imply a higher incidence of infection. however, in areas where hav is endemic, most infections occur during childhood and resolve without any lasting impact, except a robust anti-hav response 7 . fortunately, as suggested by the present study, healthy plasma donors emerge from such an area with elevated anti-hav ig titers. in fact, all plasma units from which samples for this study were pulled were negative for hav rna, an early marker of viral infection, by real-time qrt-pcr testing. it is important to note that the manufacture of ig products is globally regulated and that industry practices over the past few decades have resulted in ig products with strong pathogen safety records irrespective of the geographical region within the us from which the plasma originates. such measures include medical screening of donors, testing of plasma for disease-causing agents, and igg purification processes that incorporate segments with validated capacities to inactivate and/or remove blood-borne pathogens, in the event they were present. in the present study, plasma from six donation centers-five of which are in various locations of the us southwest-were surveyed for anti-hav ig titers. we observed a wide range of titers among the six centers, yet three obvious groups coalesced: high-titer, mid-titer, and low-titer groups. the el paso-tx and mcallen-tx centers yielded high-titer plasma with significantly higher anti-hav ig levels than those observed for all other centers surveyed. the san diego-ca and midwest city-ok centers produced mid-titer plasma, while the provo-ut center and the lone non-southwest center, clarksville-tn, exhibited low titers. we also examined if plasma from a high-titer center might also be enriched with other antiviral antibodies. similar to hav, mev and cmv are viruses for which suitable antibody levels in ig products are clinically important 22, 23 . when the full sets of plasma pools from el paso-tx and midwest city-ok were compared, there was no significant difference in anti-cmv igg levels. but the anti-mev igg level observed in midwest city-ok plasma was approximately 120% of that of el paso-tx. while the result is subtle and inverted from that of anti-hav ig, the result is interesting nonetheless. it supports the premise that regional differences in seroprevalence helps to maintain efficacious antibody levels in ig products. despite the recent decline in anti-mev titers observed for some products, current ig preparations and doses are still sufficient to provide protection against mev infection for pidd patients 24 . the analyses of individual units originating from select pools served to demonstrate that a number of centers in the us southwest are capable of consistently generating plasma with elevated anti-hav ig titers. the elevated mean titers are not singularly driven by a few donors with extraordinarily high levels; rather, the majority of individual donations exhibit values that are not more than one standard deviation above the pool mean. a similar pattern is evident in the individual donations making up pools from centers with lower anti-hav ig titers. www.nature.com/scientificreports www.nature.com/scientificreports/ furthermore, anti-hav ig titers in more than two-thirds of the individual donations from the high-titer centers were greater than the aggregate mean of plasma sample titers from the centers with lower titers. there does, however, seem to be a consistent, albeit small, number of individual donations in each high-titer pool that exhibit titers above 10,000 miu·ml −1 , which is most certainly an assuring contribution. in all, the high-titer centers consistently enrich plasma pools for anti-hav ig content. the twelve pools described in the preceding paragraph were also used to determine how differences in anti-hav ig titers relate to the capacity to neutralize hav in vitro. high-titer plasma pools from the el paso-tx center demonstrated a significantly greater capacity to neutralize hav than the plasma pools from the mid-titer midwest city-ok center, confirming that elevated anti-hav ig titers associate with greater inhibition of hav infection in vitro, which is consistent with previous observations 10 . more importantly, the neutralization result underscores the clinical benefit plasma from these high-titer centers provides. this work is timely, because, despite declining overall rates of hepatitis a infection, a spike in outbreaks of late has highlighted the need for consistent ig augmentation therapy 23, 25 . moreover, mev is reemerging as a serious public health threat with outbreaks occurring even in highly developed countries 26, 27 . notably in 2019, at the end of april the us had already seen its greatest number of reported cases since measles was said to be eliminated in 2000. meanwhile, anti-mev ig titers in the general population have also been on the decline, which has caused some ig manufacturers to have had difficulty meeting the us food and drug administration (fda) product specification for anti-mev content. to prevent shortages in product, the us fda has lowered the specification 28 . however, as a precondition for lowering the specification, the agency recommended that firms add labeling for dosing of patients needing measles pre-or post-exposure prevention. herein we report the levels of anti-hav antibodies in plasma collected from various donor centers in the southwestern quarter of the us, and we confirmed that some locales known to harbor an elevated seroprevalence produce donors with significantly higher levels of anti-hav antibodies (el paso-tx and mcallen-tx) than plasma acquired from other locations loosely associated with the us southwest (san diego-ca, midwest city-ok, and provo-ut), as well as a locale outside of the region entirely (clarksville-tn). these results are one example of how a diverse plasma supply ensures that the variety of antibodies needed for treatment of pidd is maintained. but a broader strategy deserves consideration. perhaps the production of some hyperimmune ig augmentation therapies could be made more efficient and cost-effective by a rational approach that combines epidemiologic surveillance and strategic management of the plasma supply chain rather than blind screening or immunization methods. at the very least, manufacturers of ig products could attempt to understand regional differences in antibody potency stemming from natural immunity versus immunization. on a global scale, regional sourcing has the potential to be an inherent first-line defense against emerging infectious diseases in other parts of the world. regardless of the specifics, the power of such a strategy will derive from the dynamic antibody repertoire of a diverse plasma collection platform. data reported in this manuscript are available within the article. the datasets generated and/or analysed during the current study are available from the corresponding author upon reasonable request. the epidemiology of virus transmission by plasma derivatives: clinical studies verifying the lack of transmission of hepatitis b and c viruses and hiv type 1 ensuring the biologic safety of plasma-derived therapeutic proteins effects of social, environmental and economic factors on current and future patterns of infectious diseases hepatitis a virus antibodies in immunoglobulin preparations incidence of hepatitis a in the united states in the era of vaccination hepatitis a in the era of vaccination progress toward eliminating hepatitis a disease in the united states hepatitis a antibody titres after infection and immunization: implications for passive and active immunization seroprevalence of hepatitis a virus antibodies in the u.s.: results from the national health and nutrition examination survey evaluation of potencies of immune globulin products against hepatitis a solution for intramuscular injection prescribing information, grifols therapeutics, llc human) supporting documents, new recommendations to increase the dose of gamastan s/d (immune globulin [human]) when used for prophylaxis for hepatitis a measles-virus-neutralizing antibodies in intravenous immunoglobulins prevention of hepatitis a through active or passive immunization: recommendations of the advisory committee on immunization practices (acip) rising hepatitis a immunity in u.s. military recruits infectious disease morbidity in the us region bordering mexico use of intravenous immunoglobulins for prophylaxis or treatment of infectious diseases history of immunoglobulin replacement an update on the use of immunoglobulin for the treatment of immunodeficiency disorders use of intravenous immunoglobulin in human disease: a review of evidence by members of the primary immunodeficiency committee of the american academy of allergy, asthma and immunology update on the use of immunoglobulin in human disease: a review of evidence seroprevalence of measles, mumps, rubella and varicella antibodies in the united states population official cdc health advisory distributed via the cdc health alert network (han): outbreak of hepatitis a virus (hav) infections among persons who use drugs and persons experiencing homelessness measles antibody trough levels after treatment with immunoglobulin products and predicted levels assuming lower measles antibody specifications increase in hepatitis a virus infections-united states the re-emergence of measles in developed countries: time to develop the next-generation measles vaccines? vaccine measles-united states letter to immune globulin (human) licensed manufacturers: option to lower lot release specification for required measles antibody potency testing the authors would like to thank laneisha farrar and jennifer carter for their tireless efforts to conduct the hundreds of elisas and neutralization assays necessary to complete this study. we also thank joann hotta and shih-fong chao for their expertise involving cmv propagation and neutralization. lastly, we thank mangkey bounpheng for her insightful contributions to our discussion of donor center locations. editorial assistance was provided by maryjane silvey, writemonitor, llc, durham, nc, usa, which was funded by grifols. j.c. -contributed to the study conception, design, and writing of the manuscript; was primarily responsible for data collection and analysis, as well as creation of the figures for the manuscript. k.s. -contributed to the study conception, design, and writing of the manuscript; was primarily responsible for material preparation. t.g. -contributed to the study conception, design, and writing of the manuscript. p.v. -contributed to the study conception, design, and writing of the manuscript. b.g. -contributed to the study conception, design, and analysis; was primarily responsible for writing of the manuscript. all authors are employed by grifols, the manufacturer of immunoglobulin products including gamunex ® -c, flebogamma ® dif, xembify ™ and gamastan ® . funding was provided by grifols, research triangle park, nc correspondence and requests for materials should be addressed to b.d.g.reprints and permissions information is available at www.nature.com/reprints. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-294585-dl5v9p50 authors: klein, h. g.; bryant, b. j. title: pathogen‐reduction methods: advantages and limits date: 2009-02-13 journal: isbt sci ser doi: 10.1111/j.1751-2824.2009.01224.x sha: doc_id: 294585 cord_uid: dl5v9p50 pathogen‐reduction (inactivation) provides a proactive approach to reducing transfusion‐transmitted infection. pathogen‐reduction technologies have been successfully implemented by plasma fractionators resulting in no transmission of human immunodeficiency, hepatitis c, or hepatitis b viruses by us‐licensed plasma derivatives since 1987. fractionation technologies cannot be used to treat cellular blood components. although blood donor screening, deferral and disease testing have drastically reduced the incidence of transfusion‐transmitted diseases, the threat of new or re‐emerging pathogens remains. of particular concern is the silent emergence of a new agent with a prolonged latent period in which asymptomatic infected carriers would donate and spread infection. the ultimate goal of pathogen‐inactivation is to reduce transmission of potential pathogens without significantly compromising the therapeutic efficacy of the cellular and protein constituents of blood. the acceptable technology must not introduce toxicities into the blood supply nor result in neoantigen formation and subsequent antibody production. several promising pathogen‐inactivation technologies are being developed and tested, and others are currently in use, but all of them have limits. pathogen‐reduction promises an additional ‘layer of protection’ from infectious agents and has the potential to impact the safety of blood transfusions worldwide. the first decade of the 21st century remains an age of emerging and re-emerging pathogens that threaten the blood supply. blood collectors appreciate the dramatic reduction in risk of transfusion-transmissible infections, even as they are sobered by the failures of the 20th century safeguards to prevent widespread transmission of human immunodeficiency virus (hiv) and hepatitis viruses [1] . the specter of a new, as yet undiscovered agent with an extended latent phase raises the concern that the current system of overlapping safeguards that protects patients from infectious blood components is still disturbingly vulnerable. until recently, the approach to blood safety depended upon a combination of donor education, screening, testing for selected agents and discarding components in inventory if donor exposure or illness was reported post-donation. this strategy, while effective, is reactive. pathogen reduction of blood components represents a proactive approach to blood safety [2] . inactivation technologies promise an additional layer of protection both from infectious agents that are known as well as from those not yet recognized as threats to the blood supply (table 1) . a method with broad antimicrobial activity could eliminate emerging agents before they become recognized as transfusion-transmitted pathogens. however, because blood contains numerous labile proteins and fragile cells, and because there is a wide array of potentially infectious agents, no single method of pathogen-inactivation will likely preserve all blood components, yet effectively remove all viruses, bacteria, spores, protozoa and prions. furthermore, any chemical or physical process applied to blood must be 'safe' or at least less toxic to recipients than the infectious risk of blood. many pathogens that have the potential to invade the blood supply are not yet screened by testing because of low prevalence in the general population, unknown transmission rate of infection through transfusion or the lack of a readily available test for the agent (table 1) . agents with the potential to infect the blood supply are numerous and include the known viral pathogens -more than 35 arboviruses such as the flaviviruses den-1 through den-4 and st louis encephalitis virus; the togaviruses western and eastern equine encephalitis and chikungunya; the coronavirus severe acute respiratory virus; the circovirus tt and its variant sen; and the deltavirus hepatitis d. other blood-borne viruses include the herpes viruses such as the epstein-barr virus and human herpes viruses-6, -7 and -8, as well as the human parvovirus b19 virus. numerous animal blood-borne agents (zoonotics) have been able to cause infection across species barriers; most do not cause disease, but have the potential to do so. of particular concern are the simian viruses such as the foamy viruses and sv 40 that have been reported in animal handlers and in some vaccine recipients [3] . protozoa that threaten the blood supply include the four malarial, babesia microti , found primarily in the northeastern usa, toxoplasma gondii , the causative agent of toxoplasmosis, leishmania donovani , and numerous other subspecies that result in a high disease burden in the developing world [4] . borrelia burgdorferi , the cause of tick-borne lyme disease, has the potential for blood transmission and another tick-borne illness, human granulocytic ehrlichiosis has been reported from transfusion-transmission of the bacterial pathogen anaplasma phagocytophilum [5] . four instances of transfusion transmission of the infectious protein or prion pr sc have been reported in the uk, at least three of which almost certainly caused the human equivalent of 'mad cow disease', variant creutzfeldt-jakob disease [6] . there is no blood screening test for the prion diseases. most of the world does not have access to safe blood [7] . most developing countries do not screen donor units for all viral markers, because the technology is sophisticated, the cost prohibitive and/or the incidence of infected individuals so high that little blood would be available if all markerpositive donors were excluded. approximately 50% of blood donations in developing nations are from family members or paid donors [8] . each year, unsafe blood transfusions in third world countries result in an estimated 8-16 millions hepatitis b virus (hbv) infections, 2·3-4·7 million hepatitis c virus (hcv) infections and 80 000 to 160 000 hiv infections. pathogen-reduced blood could have a dramatic impact on blood safety in these circumstances. in 1946, edwin cohn introduced what has become the most widely used commercial fractionation method for plasma proteins involving multiple steps of precipitation and physical separation by centrifugation or filtration of the precipitant and effluent using changes in ph, temperature ionic strength and ethanol concentration gradients. as pathogen-reduction occurrs after different steps of precipitation and filtration in the fractionation process, proteins isolated later in the schematic were generally safer from infectious agents [9] . the albumin and globulin fractions proved extremely safe, particularly regarding transmission of hepatitis viruses and hiv. however, the process was not infallible and deviations from proper processing have resulted in disease transmission. some of the most important plasma protein derivatives such as factors viii and ix are separated early in the fractionation process and do not reap the benefits of added layers of fractionation and processing. pasteurization has been used effectively to inactivate viruses in albumin fractions stabilized with small chain fatty acids from as early as 1948 [10] . other proteins, especially clotting factors, denature during the pasteurization process unless additional stabilizers are added. terminal heat inactivation of lyophilized clotting factors has brought varied results depending upon temperature and processing time. dry heat treatment of lyophilized clotting factors at 60-68 ° c does not prevent transmission of hbv, hcv and hiv. increasing the dry heat temperature to 80 ° c for 72 h destroys hiv, hbv and hcv, although the non-enveloped viruses, especially hav and human parvo b19, may not be completely inactivated. adding humidity (vapor heating) improves viral kill. heat inactivation at 100 ° c for as little as 1 h has resulted in viral inactivation of both lipid enveloped and non-enveloped viruses. when this process is applied to intravenous immunoglobulin concentrates, little protein is lost. exposure to low ph inactivates many enveloped viruses, however among commercial proteins, only the immunoglobulins are stable under the necessary acid conditions (ph 4·0). use of organic solvents and detergents in the processing of coagulation factors inactivates the lipid-enveloped viruses, but not the non-lipid enveloped viruses [11] . the solvent and detergent combination disrupts the lipid envelope and prevents the virus from binding to cells and replicating. incorporation of a virucidal detergent and solvent (solvent 1% tri-n-butyl phosphate and the detergent 1% triton-x-100 for 4 h at 30 ° c) into the processing of plasma from pools of approximately 12 000 donors produces a product known as solvent-detergent plasma [12] . the tri-n-butyl phosphate is removed by oil extraction, and triton-x-100 is removed by chromatographic adsorption. plasma protein concentrates have been made from solvent-detergent-treated plasma, and fresh-frozen plasma (ffp) equivalent has been licensed in the usa and europe. as with the heat-inactivated coagulation factors, solvent-detergent technology does not inactivate the nonenveloped viruses. solvent-detergent treatment also results in some loss of integral plasma proteins such as α -2 antiplasmin and protein s [12, 13] . alpha-2 antiplasmin is crucial in maintaining haemostasis especially in patients with liver dysfunction. decreased levels of the natural anticoagulant protein s can lead to a hypercoaguable state especially when massive solvent-detergent plasma transfusions are used as in massive traumas or therapeutic plasma exchanges. these concerns, along with economic factors, resulted in the removal of solvent-detergent plasma from the us market. solvent-detergent plasma is still used widely in europe. recently, two new solvent-detergent treatment procedures have been developed for single unit or mini-pools of 10-12 units of plasma that yield > 90% mean recovery of coagulation factors, anticoagulants (including protein s), protease inhibitors (including α -2 antiplasmin), total protein, albumin and immunoglobulins. single unit and mini-pool solventdetergent treatment technologies show promise and have the potential to overcome some of the drawbacks of the original industrial solvent-detergent treatment processes [14] . nanofiltration has proven effective in removing a wide range of viruses including the non-enveloped viruses, and may even remove viruses smaller than the filter pore size [15] . many currently licensed plasma derived coagulation factors and immunoglobulins that are subjected to heat, pasteurization and/or solvent-detergent treatment are also nanofiltered. all classes of plasma protein fractions such as antithrombin, c-1 inhibitor, protein c, fibrinogen and ceruloplasmin have been nanofiltered without apparent change in the protein characteristics. methylene blue (mb) is a photoactive phenothiazine dye that has been used in europe for more than 15 years for the pathogen-inactivation of single units of plasma. mb has an affinity for nucleic acids and the surfaces of viruses [16] . when mb-treated plasma is exposed to ultraviolet light, most enveloped viruses are easily inactivated; however nonenveloped viruses are more resistant. intracellular viruses are not inactivated by mb/ultraviolet light, but freezing and thawing plasma often disrupts the cell membranes of leucocytes, thus liberating the viral particles and leaving them susceptible to mb pathogen-inactivation. residual intact white blood cells containing viruses are removed by a micropore filter. neither protozoa nor bacteria are inactivated by mb treatment. plasma proteins are moderately affected; fibrinogen and fviii activity is reduced by up to 30% [17] . mb treatment can be used for pathogen-inactivation of single units of plasma, thus eliminating the risk of large plasma pools currently used to manufacture solventdetergent plasma. macopharma uses an in-line system consisting of a membrane filter, mb dye, illumination bag, elimination filter and storage bag. the 0·65 μ m membrane filter removes platelets, leucocytes and debris. the plasma then passes through tubing containing an mb pill that dissolves as the plasma flows through the tubing into the illumination bag. the mb-containing plasma is subjected to double-sided illumination by sodium high-intensity low-pressure lamps emitting yellow light at a wavelength of 590 nm for 15-20 min. plasma is then passed through an mb elimination filter that removes greater than 95% of the residual dye and photoderivative by-products [18] . millions of mb single unit of plasma have been transfused in europe without unexpected adverse outcomes. until recently, attempts at pathogen-reduction for cellular blood components have achieved little success. leucoreduction of blood has reportedly decreased the risks of transfusion transmitted cell-associated viruses, such as cytomegalovirus, human t-lymphotropic virus i/ii and probably epstein-barr virus and human herpesvirus-8 (hhv-8), as leucocytes are the principal reservoir for these infectious agents. psoralens are small, planar molecules that cross cell membranes and viral capsids and intercalate between the bases of the nucleic acids. upon illumination with ultraviolet a (320-400 nm), the psoralens react with the dna or rna pyrimidine bases to form covalently bonded intranucleic and internucleic acid cross-links. this cross-linking prevents replication and transcription of the rna or dna [19] . psoralen treatment with ultraviolet a light results in the reduction of a broad array of viruses, bacteria and protozoa to a level unlikely to transmit infection. aminomethyl-trimethyl psoralen, a three-ringed synthetic psoralen known as amotosalen hydrochloride or s-59, has been extensively tested in the pathogen-reduction of platelets and plasma. amotosalen and photochemical treatment have demonstrated an acceptable safety profile through extensive toxicological studies for acute toxicity, repeat dose toxicity, reproductive toxicity, phototoxicity, and mutagenic and carcinogenic potential. in order to pathogen-inactivate with psoralens, the platelet concentrate must be volume reduced and re-suspended in 30-45% plasma and 70-55% platelet additive solution. the amotosalen is added to the platelet and incubated for 3-5 min [20] . the product is then exposed to ultraviolet a light after which approximately 80% of the psoralen have been photodegraded to by-products. the remaining psoralen and by-products are removed by a 'compound absorption disc' . intercept, an s-59 amotosalen system, has been evaluated in three clinical trials in europe (eurosprite) involving 166 thrombocytopenic patients. two of these trials evaluated whole blood-derived buffy-coat platelets, and one assessed single donor apheresis platelets. these studies demonstrated that when equal platelet doses were transfused, the inter-cept and conventional platelet transfusions resulted in comparable post-transfusion platelet count increments without significant differences in adverse reactions. in the usa, the sprint trial evaluated the haemostatic efficacy and safety in 645 thrombocytopenic oncology patients receiving intercept single donor apheresis platelets collected on the amicus separator [20] . a total of 4719 platelet transfusions were given; 2678 intercept platelets and 2041 conventional platelets. the incidence of world health organization grade 2 bleeding between the groups was comparable, and the incidence between the groups of world health organization grade 3 or 4 bleeding was equivalent. patients receiving the intercept platelets had lower post-transfusion platelet count increments, required more platelet transfusions and had a shorter interval between transfusions. explanations for the differences in post-transfusion platelet count increments in the photochemical-treated (pct) platelets were partly explained by the lower mean platelet dose and disproportionate number of transfusions containing platelet doses less that 3·0 × 10 11 cells. transfusion reactions were fewer with pct platelets most likely attributed to the reduced volume of plasma in the pct units as well as increased leucocyte inactivation resulting in less cytokine production during storage [21] . however a trend towards a higher frequency of respiratory complications has been noted and is of great concern to the us food and drug administration, but apparently less so for other national regulatory bodies. amotosalen and ultraviolet a light has been used to pathogeninactivate plasma in a system much like that used for platelets [22] . post-thaw coagulation studies of the pct plasma demonstrate that most coagulation factors are well-preserved in the range of 73-98% of control thawed plasma. factor viii levels, while decreased to 73%, are still sufficient for therapeutic use. there were no significant differences in the quantity and activity of the von willebrand factor, the pattern and distribution of the von willebrand multimers, or the activity of the adamts-13. fibrinogen maintained functional activity of approximately 87%. protein c and s as well as antithrombin were maintained at > 95% pre-treatment activity levels. there was no evidence of coagulation factor activation as a result of treatment. the factor vii kinetics of post-transfusion pct plasma was compared to standard ffp in a crossover study [23] . in a study of 34 patients with congenital coagulation factor deficiencies, coagulation factor kinetics and therapeutic efficacy of ffp treated with amotosalen and ultraviolet a light has been shown to be consistent with that of conventional ffp [24] . randomized controlled trials of pct-ffp supported haemostasis for the treatment of acquired co-agulopathy of liver disease and liver transplantation have revealed outcomes similar to that of conventional ffp [25] . additional studies utilizing pct-ffp for plasma exchanges in thrombotic thrombocytopenia purpura demonstrated similar results as those with conventional plasma. cryoprecipitate can also be produced from amotosalen and ultraviolet a-treated plasma. preliminary studies indicate that pct cryoprecipitate coagulation factor levels are acceptable. riboflavin, vitamin b2 a naturally occurring essential nutrient, has been used as a pathogen-inactivating agent for platelets and plasma. riboflavin is a 3-ringed planar structure that binds to nucleic acids and intercalated between dna and rna bases. upon activation of cross-linked riboflavin with ultraviolet or visible light, guanosine bases are oxidized resulting in single strand breaks in the nucleic acids. the damaged and disrupted nucleic acids are incapable of repair and replication. toxicities of riboflavin and its photoderivative by-products do not appear to cause concern, because riboflavin and its breakdown products are present in many food and natural products. removal of the spent riboflavin and products post-illumination may not be necessary in a pathogen-inactivation system using riboflavin. the us food and drug administration has classified riboflavin as a 'generally regarded as safe' compound. the mirasol prt system contains 30 ml of riboflavin (500 μ m) in a light protective pouch and a pathogen-reduction illumination/storage bag. platelets or plasma are sterilely connected to the system, and 250 ml of plasma or platelet product is transferred to the bag containing the riboflavin diluting it to a final concentration of 50 μ m. the riboflavin-treated product is subjected to double-sided ultraviolet illumination [27] . riboflavin/ultraviolet light treatment has been evaluated in preclinical studies and found to result in reduction of infectivity by many pathogens including west nile virus, intracellular hiv, bacteria and protozoa. the mirasol system demonstrated successful pathogen reduction of selected pathogen-spiked platelet units after treatment and storage for 5 days. the viral reduction of platelets was sufficient to close the window period of transmission of hiv and chronic phase of parvo b19, eliminate the viraemic period of west nile virus, prevent infection due to staphylcoccus epidermidis and escherichia coli , and result in a 5-6 log reduction of leishmania donovani infantum [27] . additionally, leishmaniaspiked plasma units treated with riboflavin/ultraviolet light demonstrated a 5-7 log reduction in parasites. studies have demonstrated significant differences in control and treated platelets after 5 days of storage in regards to accelerated changes in platelet morphology, increased platelet activation and induced partial platelet aggregation. riboflavin/ultraviolet light-treated plasma has been shown to retain acceptable levels of clotting factors without evidence of increased compliment activation. pathogen-inactivation of components containing red blood cells presents a particularly challenging dilemma. methods utilizing photoactivation must do so in the red wavelength region of the light spectrum above that of haemoglobin in order to avoid absorption or scattering of the light by the red blood cell. many potential methods of pathogen-inactivation easily alter or disrupt the red blood cell membrane resulting in decreased red cell survival, haemolysis or immunogenicity. s303 (helinx), a small molecule designed for pathogeninactivation treatment of red blood cells, is an alkylating agent derived from a quinacrine mustard that belongs to a class of 'frangible anchor linker effectors' (frale) compounds. frale compounds contain an intercalator group that inserts into the helical region of dna and rna, an effector group that permits covalent attachment of nucleic acids and a central frangible bond that orchestrates the degradation of the compound [28] . s-303 is a positively charged planar structure that easily intercalates into the helical regions of the negatively charged nucleic acids. the process does not depend on light for activation. frale compounds are activated by a shift from lower ph storage environment to the higher neutral ph of red blood cells causing hydrolysis and generating s-300 the primary degradation product; cross-linking of the dna and rna ensues. s-300 is rapidly metabolized and excreted leaving no detectable parent compound. the remaining free degradation products are absorbed and removed by a compound removal step. s-303 binds to other proteins and cell membranes as well as nucleic acids, and up to 20% can potentially remain bound to the surface or contained within the red blood cells. s-303 has demonstrated pathogen-inactivation of a wide range of viruses, bacteria and protozoa. no unexpected toxicities have been described. assays for red blood cells storage lesions (extracellular potassium leakage, plasma-free haemoglobin, adenosine diphosphate, 2,3-diphosphoglycerate, glucose and lactate) are comparable to control red blood cells. the red blood cell function appears to be normal, and in vivo 51 cr-labelled survival studies exceed the standard of 75% at 24 h. two randomized, controlled trials involving patients either undergoing first-time cardiovascular surgery or with haemoglobinopathies were in progress when antibodies to residual red blood cell bound s-303 were discovered in two subjects [29] . the trials were suspended as a consequence of these findings. additional studies have revealed that 1% of patients and healthy donors that had never been exposed to s-303 had naturally occurring antibodies that reacted with s-303 treated red blood cells. modifications have been made to the s-303 treatment process to reduce the amount of red blood cell bound s-303 in attempts to eliminate immunoreactivity and immunogenicity. preliminary finding indicates that red blood cells from the modified s-303 treatment process were cross-match-compatible with the anti-s-303 antibodies formed after exposure to the original s-303 formulation as well as the anti-s-303 antibodies found in the patients and donors never exposed to s-303. the antibodies do not appear to impair transfusion or to pose any clinical problem. new clinical trials have been initiated. riboflavin-based pathogen-inactivation systems for red blood cells are currently under development if found to be a successful means of pathogen-inactivation of red blood cells, riboflavin may serve as the one material to inactivate pathogens in three blood components (red cells, platelets and plasma). current leucoreduction filters are effective in removing cell-associated viruses, but remove only 42% of total prion infectivity in endogenoussly infected blood. a leucoreduction filter under development removes prions in exogenously and endogenously infected blood more effectively [30] . exogenous infectivity studies were conducted using 270 ml of red blood cells and 30 ml of 10% (wt/vol) with high titre brain homogenates from hamsters infected with scrapie for a final 1% homogenate concentration. endogenous infectivity studies utilized red blood cells processed from 500 ml of whole blood collected from 120 scrapie-infected hamsters. the new prototype leucoreduction, prion reduction filter was effective in removing 3·7 logs of scrapie infectivity from exogenously infected red blood cells and all of the detectable prp sc from the endogenously infected hamsters. in the endogenous infectivity study, the pre-filtered-infected red blood cells transmitted disease to six of 43 animals, and the post-filtration red blood cells did not transmit disease to any of 35 animals. allogeneic blood is a critically important therapeutic, but also an inherently risky biologic source material. among the dangers is transmission of a wide range of pathogens. donor selection and blood testing have reduced this risk dramatically and will remain the cornerstone of blood safety programmes. nevertheless, infectious units still elude screening and testing; testing errors and product release errors are probably impossible to eliminate. the largest current infectious risks involve pathogens for which we do not test, for which we have no test, or for which demographic screening and testing are inadequate. the greatest fear concerns the emergence of a 'new' transmissible agent, particularly one that has not previously been associated with human disease, has a long silent period, can infect others by secondary spread and is highly lethal -as was the case with hiv. ideally, pathogeninactivation techniques would provide an additional safeguard. that has been the experience in the plasma fractionation industry. no single pathogen-reduction method will likely be effective for every class of agent and for every blood component. some combination of techniques that remove and inactivate infectious agents will probably be needed. these technologies are generally expensive and have the potential to profoundly escalate the cost of blood, but this cost may be partially offset by the elimination of some testing markers. however, if potent pathogen-inactivation techniques that preserve blood function and do not evidence some new toxic risk can be created, the developed world, which embraces the myth of zero-risk transfusion, will likely adopt them almost regardless of cost. for the developing world, in which low-risk blood donors are at a premium and elegant testing methods often not feasible, a good but not perfect pathogen reduction method, especially if relatively inexpensive and easy to implement, could save millions of lives. meeting transfusion safety expectations pathogen inactivation: making decisions about new technologies. report of a consensus conference frequent simian foamy virus infection in persons occupationally exposed to nonhuman primates protecting the blood supply from emerging pathogens: the role of pathogen inactivation risk and prevention of transfusion transmitted babesiosis and other tick-borne diseases preclinical vcjd after blood transfusion in a prnp codon 129 heterozygous patient who: fact sheets: blood safety and volunteer donations blood supply and demand clearance of prions during plasma protein manufacture an adventure in biotechnology: the development of haemophilia a therapeutics -from whole blood transfusion to recombinant dna to gene therapy sterilization of hepatitis and htlv-iii viruses by exposure to tri (n-butyl) phosphate and sodium cholate current status of solvent/detergenttreated frozen plasma venous thromboembolism associated with the management of acute thrombotic thrombocytopenic purpura a process for solvent/detergent treatment of plasma for transfusion at blood centers using a disposable bag system removal of small non-enveloped viruses by nanofiltration virus inactivation in blood components by photoactive phenothiazine dyes methylene blue treated fresh-frozen plasma: what is its contribution to blood safety? methylene blue and thionine in pathogen inactivation of plasma and platelet concentrates photochemical inactivation of viruses and bacteria in platelet concentrates by use of a novel psoralen and long-wavelength ultraviolet light therapeutic efficacy and safety of platelets treated with a photochemical process for pathogen inactivation: the sprint trial clinical safety of platelets photochemically treated with amotosalen hcl and ultraviolet a light for pathogen inactivation: the sprint trial preclinical safety profile of plasma prepared using the intercept blood system pharmacokinetic study of ffp photochemically treated with amotosalen (s-59) and uv light compared to ffp in healthy volunteers anticoagulated with warfarin fresh frozen plasma prepared with amotosalen hcl (s-59) photochemical pathogen inactivation: transfusion of patients with congenital coagulation factor deficiencies photochemically treated fresh frozen plasma for transfusion of patients with acquired coagulopathy of liver disease photochemical inactivation of selected viruses and bacteria in platelet concentrates using riboflavin and light the use of riboflavin for the inactivation of pathogens in blood products helinx technology for inactivation of infectious pathogens and leukocytes in labile blood components: from theory to clinical application therapeutic efficacy and safety of red blood cells treated with a chemical process (s-303) for pathogen inactivation: a phase iii clinical trial in cardiac surgery patients removal of exogenous (spiked) and endogenous prion infectivity from red cells with a new prototype of leukoreduction filter key: cord-270908-9snyt2n1 authors: persson, c. g. a.; andersson, m.; greiff, l.; svensson, c.; erjefält, j. s.; sundler, f.; wollmer, p.; alkner, u.; erjefält, i.; gustafsson, b.; linden, m.; nilsson, m. title: airway permeability date: 2006-04-27 journal: clin exp allergy doi: 10.1111/j.1365-2222.1995.tb00022.x sha: doc_id: 270908 cord_uid: 9snyt2n1 nan the upper airway mucosa is a main site for deposition of potentially noxious environmental molecules. as one might expect this mucosa is also well equipped to protect both itself and the rest of the body from harmful influences of foreign material [1] . the regulation of the permeability of nasal and tracheobronchial mucosa is such that blood plasma may enter the airway lumen without making it any easier for foreign surface molecules to penetrate into the airway tissue [2] [3] [4] . thus, circulating humoral defence systems would be allowed to neutralize offending stimuli on the surface of a mucosa that maintains its absorption barrier uncompromised [5] . mechanisms involved in the direction-selective paracellular flux of solutes across the intact airway mucosa [6, 7] are the basis for the first part of this overview. if the inhalational insult or disease process are severe enough for epithelial lining cells to be shed, even this may occur without a deleterious loss of barrier function. if basal cells remain they may promptly flatten out and establish cell to cell contacts [8] . if both columnar and basal cells are removed a provisional plasma-derived gel immediately covers the denuded basement membrane [9] . furthermore, beneath the gel restitution of a new epithelium proceeds speedily [10] . these novel aspects on barrier restitution after shedding are the basis for the second part of the present overview. most of the data that will be discussed have been generated in the human nose and the guinea-pig trachea. the focus is primarily on in vivo observations. explanatory or expanding ex vivo findings have thus been included when the functional in vivo aspects have been first assessed. during the first world war felix marchand published his pioneering work on similarities of pathological features in nasal and tracheobronchial mucosa in asthma correspondence: c. persson, department of clinical pharmacology, university hospital, s-221 85 lund, sweden. [11] . he focused on a role of eosinophils, mast cells, and epithelial cells in this disease. marchand argued against the notion, previously forwarded by fraenkel [12] , that denudation characterizes asthmatic airways. he further regarded the epithelial lining cells not only as a passive barrier but as effector cells potentially driving inflammatory processes. there is now a resurgence of interest in versatile proinflammatory roles of the airway epithelium [13, 14] including allergen presentation pathways and subsequent t cell mechanisms which may confer lifelong allergic reactivity [15] . these are important aspects on the airway 'barrier cells'. however, they are outside the scope ofthe present discussion. in the beginning of this century asthma and rhinitis were considered for the first time to be allergic diseases [16] . as a corollary there was then a rapidly growing interest in mucosal penetration of inhaled allergenic substances. it was soon realized that the normal airway-alveolar mucosa would absorb even large allergen molecules. the first specific airway absorption studies involved experiments in the nose [17] avoiding any contribution of alveolar-pulmonary absorption. (the latter route is difiicult to distinguish from tracheobronchial absorption in studies involving the lower airways.) authors interested in allergic and occupational airway diseases have continued to study nasal absorption in human subjects. based on select human nasal and animal airway observations, in the 1960s and onwards, the paradigm was developed that the airways of atopic individuals are characterized by 'excessive mucosal permeability' allowing abnormal penetration of inhaled molecules [18] [19] [20] [21] [22] . despite its widely acknowledged attractiveness this notion may now need to be thoroughly re-evaluated. for example, several apparently supporting observations may require reinterpretation. the common presence of plasma proteins on airway mucosal surfaces in asthma and rhinitis has been thought, wrongly as we now see it [5] , to be a clear sign of a general hyperpermeability state. absorption data obtained under less well controlled conditions [23] have been generously interpreted in favour of the hyperpermeability hypothesis or, when failing to support the accepted paradigm [24, 25] the data may not have received widespread attention. although not quantitatively confirmed, histological pictures have been publicized showing all the features of the paradigm of hyperpermeability in human allergic airways, including paracellular epithelial 'gaps' in the nasal mucosa [26, 27] and denuded basement membranes in asthmatic bronchi [28] . as a rule the reductive molecular and cellular approaches in medical research are strictly dependent on established paradigms concerning the gross physiological functions in complex in vivo biosystems. hence, it is not surprising that intriguing molecular mechanisms, explaining how the epithelial absorption barrier would become deranged in allergic airway disease, have appeared and received support from in vitro cell and cell culture observations [29] [30] [31] . perhaps the notions have developed differently had the early work by cohen et al. [24] been remembered. using the prausnitz-kustner reaction to assess nasal mucosal absorption of allergen cohen et al. [24] noted that absorption was much faster and more efficient in healthy subjects than in rhinitis; the poorest absorption rates occurred in those patients who developed an allergic reaction to the topically applied allergen. recent findings on airway absorptivity in allergic disease confirm and extend the data reported by cohen et al. 65 years ago. it now seems possible that longstanding exudative, eosinophilic mucositis can be associated with an airway absorption barrier that is even tighter than in health [32] . the current work on the function of the airway absorption barrier is multifaceted. there is interest in the use of nasal application and oral inhalation for systemic absorption of drugs such as peptides, which cannot be ingested because they are destroyed by gastrointestinal juices. this area of research also includes the study of a variety of absorption enhancer moleculesformulations. endogenous and environmental mechanisms potentially involved in development of frailty and detachment of airway epithelial cells also continue to be examined. the focus is on causative factors such as toxic proteins, oxidants, and proteases as well as physical hydrostatic and osmotic forces. eosinophils and neutrophils are considered the most important epitheliumdamaging effector cells. the vast interest in epithelial damage and shedding refiects the multipotential importance of the airway epithelium in health and in disease processes. however, we think it is equally important to focus the discussion on the epithelial repair processes that ought to be ongoing in asthma and rhinitis. recent observations in guinea-pig trachea in situ have unravelled some intriguing aspects of the mucosal repair functions. erjefalt et al. [9, 10] have thus demonstrated that a denuded airway basement membrane immediately receives a provisional cover that emanates from the microcirculation and that restitution of an epithelial lining may occur exceedingly fast beneath this cover. epithelial disruption and shedding, even denudation, may, therefore, not be that detrimental to the airway barrier function as we have been inclined to believe. topical application of histamine-type mediators (histamine, leukotriene d4, bradykinin, platelet activating factor [paf]) on the airway mucosa results in immediate extravasation and luminal entry of plasma. an identical acute response is produced by topical allergen in sensitized subjects or in animals [33] . the plasma that is moved into the airway lumen may not have been much sieved. small and large proteins, including q:2-n^acroglobulin, thus enter the lumen at concentration ratios approaching those existing in the circulation. however, the plasma exudate is distinct from circulating plasma by its rapidly increasing content of protein breakdown products. the plasma-derived peptides and oligoproteins are not inert. bradykinins, complement fragments, and fibrinolysis peptides belong to those relatively few plasma-derived molecules whose actions have been partly elucidated. plasma proteins may avidly bind interstitial molecules of the airway mucosa. subepithelial cytokines and other cell-derived mediators may thus be picked up and transported to the airway surface by the plasma exudation process. this possibility has been termed 'lamina propria lavage' [34] . luminal mediators may emanate directly from activated superficial cells in the airways or they may emanate from subepithelial sources and merely be moved to the surface by an induced plasma exudation response (for example by an allergen challenge procedure). speculatively, at ongoing airway exudation processes the surface may accumulate and the tissue may be depleted of such cellular release products that bind to plasma proteins. the luminal entry of bulk plasma is a graded response. this aspect has been examined particularly in the immediate phase (usually within 10-20 min after challenge). the stronger the topical stimulus the more plasma is exuded per unit time. the threshold dose levels of exudative agents that brings about only marginal increases in tissue plasma still produces clear increases in airway luminal plasma [33] . hence, luminal entry is a rapid and efficient clearance route for extravasated tissue plasma. lymphatic removal may only have a marginal role at least at exudation responses evoked by acute airway challenges [35] . this also means that the plasma indices of surface samples may accurately assess an airway permeability 809 increased permeabihty response in subepithehal microvessels. this latter aspect is important because it makes it relatively easy to properly examine the microvascular responsiveness to different challenges. also, the lack of luminal plasma proteins after challenge with neurogenic agents such as capsaicin and nicotine can now be taken as conclusive support for the notion that neurogenic inflammation (plasma extravasation) may not occur in human airways [33] . the human nasal mucosa lends itself to airway specific, well controlled challenge and lavage studies. to take advantage of these possibilities a nasal pool technique has been developed [36] . using a compressible nasal pool devise it is possible to fill the entire ipsilateral nasal cavity with fluid and solutes that will not be removed by any ciliary activity. the pool technique exposes a large airway mucosal surface area to defined concentrations of agents and tracers. the technique also allows exposure of the same airway mucosal surface area at repeated provocations. after a selected mucosal exposure time the pool fluid, almost quantitatively, is recovered into the device. thus the exposed mucosal surface is also gently lavaged by the nasal pool fluid providing the opportunity to sample mucosal indices selectively from the area of interest. this gentle lavage procedure can be carried out numerous times in sequence without causing undue changes in mucosal function. it has not been possible to attain similarly controlled experimental conditions in human trancheobronchial airways. however, it is possible that findings in the nose on mucosal functions may be applicable also to the lower airways [33] . using the nasal pool device greiff et al. [36] have demonstrated graded exudative eflects of difl"erent mucosal surface concentrations of histamine. between 20 /xg/ml and 2000 /ig/ml this amine produces fivefold to more than 100-fold increases in lavage fluid levels of plasma proteins (albumin to a2-inacroglobulin). it appears that bulk plasma exudate with all its large proteins and active products is transmitted to the mucosal surface without any appreciable disturbance of the epithelial lining. several experiments involving human and animal airways have thus demonstrated that the acute plasma exudation response produced by histamine-type mediators or allergen is not associated with any change in the ability of the airway mucosa to absorb small and large hydrophilic solutes. the airway mucosa displays a remarkable asymmetry in that a dramatic increase in the 'outward permeability' occurs without any concomitant or subsequent change in the 'inward permeability' [2] [3] [4] [5] . these observations have led to the identification of a role of plasma exudates in airway surface defence also when the mucosa is normally intact [5] . from a defence point of view it seemed logical that the epithelial lining should allow the plasma solutes to reach the surface without causing epithelial damage and without loss of the normal mucosal barrier functions. however, what properties ofthe epithelium and what external influence could produce such a result? the cumulating in vivo observations indicate that actual secretion may not be involved in the luminal entry of plasma [37] . although in vitro studies have suggested the albumin may be actively secreted by airway mucosal tissue, this possibility has not been born out in vivo. the in vitro secretion comprises only albumin. other macromolecular tracers that move along with albumin in the exudation process are not secreted. the in vitro secretion mechanism and the in vivo exudation of plasma are also entirely differentiated by agents which stimulate one and not the other, and by agents that stimulate one and inhibit the other [37] . as a matter of fact, it never occurred to us that secretion could be involved because we had observed that bulk plasma comparable to the collective volume of the cytoplasm ofthe epithelial lining cells could enter the airway lumen in a few minutes. the only conceivable pathways for this transmission would be extensive paracellular routes. the pattern of response produced by difl"erent inducers and inhibitors suggested that the luminal entry of plasma was an obligatory consequence of extravasation. for example, no data suggested that the passage specifically across the epithelial linking was subjected to selective pharmacological regulation. from the in vivo findings it was thus deduced that the extravasated plasma itself somehow might create pathways for its own luminal entry [17] . physiologists, who have examined water and solute flux across gallbladder and other epithelia [38] [39] [40] , apparently have not paid attention to the possibility that macromolecular plasma exudates may move into the lumen of cavitary organs through simple and sensitive hydrostatic pressure regulated paracellular mechanisms. the focus has rather been (and still is, [29] ) on the possibility that subepithelial hydrostatic pressure and oedema could be a cause of epithelial disruption and sloughing. we formulated a novel hypothesis that could be tested by examining the influence of small increases in subepithelial hydrostatic pressure on airway luminal entry of macromolecular tracers. the test was carried out in vitro employing as undisturbed airway tissue as possible. the guinea-pig trachea was isolated as an intact tube and kept in an organ bath by means that allowed separate regulation of the levels of serosal and mucosal bathing fluids, respectively. using this model we followed the movement of fluorescein isothiocyanate labelled dextran (mw 70 000) across the mucosa under conditions when different hydrostatic pressure gradients had been created across the airway mucosa. it was exciting to see the first data showing that a hydrostatic pressure increase of merely 5 cm h2o on the serosal side significantly increased the luminal entry of macromolecules [6] . this increase was induced promptly and was rapidly reversible when the pressure load was taken away. the whole process was well repeatable at 30 min intervals and no change was produced in the epithelial structure or in solute indices such as lactate dehydrogenase that would have been increased if the epithelium had been damaged. the basolateral sides of the cylindrical epithelium of the airway mucosa is particularly sensitive, since application of a pressure load of similar magnitude on the mucosal surface produced no change in the flux of macromolecules in either direction. also, the function ofthe mucosa as an absorption barrier was unimpeded during and after the hydrostatic pressure-induced movement of macromolecules to the airway surface [7] . it was further demonstrated that several agents, that either induced or inhibited exudation in vivo, did not afl"ect the hydrostatic pressure-induced luminal entry of macromolecules in the isolated tracheal tube preparation. all these observations [6, 7] were in excellent agreement with the previously established characteristics of the airway plasma exudation process in vivo in both guinea-pigs and in human subjects. the hypothesis was advanced that extravasated bulk plasma moves non-injuriously into the airway lumen by slightly increasing the hydrostatic pressure load on the basolateral aspect ofthe epithehal lining cells. more detailed histological examination of tissues from in vivo studies indicate that the epithelial passage of bulk plasma is across abundant paracellular routes. the pictorial evidence emerging from studies with tracers of high resolving power thus suggest that plasma macromolecules move between and all around each epithelial cell with no preferred routes observed [41] . this kind of passage means that the burden on each stretch of epithelial apical connections (1 cm^ of the mucosal surface may have 40 m of interepithelial contact lines) will be exceedingly small even at pronounced plasma exudation responses. this again is well compatible with the previously established non-injurious nature of mucosal exudation of bulk plasma. the exudation data discussed above suggest a high degree of plasticity of the tight junctions at the apical pole of airway cylindrical epithelium. these junctions appear to have a valve-like function. they readily yield to small hydraulic pressures moving up between the cells and at completion of the mucosal exudation process the junctions apparently resume tightness in a fully reversible way. reactive oxygen products, surfactant active agents, occupational and other toxic chemicals may cause acute epithelial damage and thereby increase the absorption of solutes across the airway mucosa [3, 31, 33, 42] . proteases, eosinophilic proteins [30] , and certain viral infections [43] may also produce epithelial damage and thus increase the inward perviousness of the mucosa. if airway absorption of inhaled material is increased this may cause significant adverse effects because subepithelial cells and different airway end-organs will be abnormally exposed to potentially harmful environment agents. an increase in airway permeability is actually desired when systemic drugs, such as insulin and other peptides, are applied on the airway mucosa for systemic treatment purposes. to this end absorption enhancers are used [44] . however, to be acceptable for repeated topical airway application, absorption enhancers should not cause longlasting epithelial changes nor should they cause other significant airway mucosal effects. an absorption enhancer mechanism should thus involve significantly increased airway absorption for a well defined period of time without inducing significant adverse mucosal reactions. oxygen radicals as generated by h2o2, surfactantactive agents such as dioctylsodiumsulphosuccinate, water-absorbing particles such as starch beads, and sodium caprate increase absorption of hydrophilic molecules across airway mucosa [3, 42, 44, 45] . all these agents also produce plasma exudation responses [3,42, unpublished observations] . indeed, as demonstrated with a range of histamine-type mediators it appears much easier to increase exudation than absorption [5] . the intensity and duration of airways plasma exudation may quantitate such adverse mucosal processes which are of significant inflammatory nature [46] . hence, we suggest that the plasma exudation response may reflect to what extent the absorption enhancement is achieved at the expense of other less desired airway actions. if absorption enhancers are not devoid of an exudative effect this latter action should be shortlasting or it may reflect a serious adverse effect of such pharmaceutical agents. non-speciflc hyperresponsiveness in the human nose may be assessed as abnormally increased challengeinduced symptoms (blockade, 'secretion', sneezes, and irritation-pain) at challenge with methacholine or histamine [47] . by employment of proper challenge stimuli we may get additional information as to the selective responsiveness of sensory nerves, the secretory apparatus, the microcirculation, etc. [33, 47] . an increased penetration of challenge agents might equally explain non-specific and the latter specific end-organ types of airway hyperresponsiveness. but, it is doubtful whether increased penetration and absorption may apply in hyperresponsive airways. the occupational agent tdi produces longlasting airway plasma exudation responses already in animals that have not been sensitized to this agent [48] . the acute exposure to tdi also involves structural epithelial changes but only a marginal increase in the absorption of lumenal molecules. when the animals have become sensitized to tdi they respond with exudative eosinophilic inflammation to exceedingly low doses (< 1 nl/ animal) of this agent [49] . tdi-asthma-rhinitis is associated with plasma exudation and hyperresponsiveness [50] but whether this occupational disease is associated with any change in airway absorption now remains unknown. it has recently been demonstrated that the common cold viruses may produce significant disease symptoms, hyperresponsiveness, and exudation of plasma macromolecules without causing appreciable increases in the human nasal airway absorption permeability. coronavirus-induced nasal infection is thus associated with increased plasma exudation responses to histamine [51] . this disease characteristic probably reflects true changes in the responsive end-organ (microcirculation) since increased penetration of the challenge agents may not apply [51] . severe airway infections caused by human influenza virus may be associated with extensive airway epithelial damage and shedding [43] but the extent of increased mucosal absorptivity under these conditions now remains little studied. using the controlled conditions that are offered by the nasal pool technique [36] greiff et al. [32] have observed that the nasal absorption of a small hydrophilic tracer (cr ^^edta) is abnormally slow in subjects with allergic rhinitis. thus, late into the swedish birch pollen season when eosinophilic exudative inflammation would have been present for several weeks the allergic airway mucosa exhibited an increased functional tightness. in a separate study similar findings have now been obtained concerning peptide absorption across the allergic nasal mucosa [52] . during the swedish pollen season rhinitic individuals also develop a significantly increased responsiveness to histamine challenge expressed as abnormally increased plasma exudation effects [53] . since absorption of histamine may be decreased in these patients the recorded hyperrresponsiveness may be an underestimation of the change that had occurred in the airway microcirculation. it may not be feasible to have perfectly controlled conditions for studies of absorption across a defined bronchial mucosal surface in vivo in man. however, in a recent study halpin et al. [54] made serious attempts to correct for mucociliary transport of the inhaled absorption tracer and could demonstrate that the absorption permeability in asthma may be reduced. it appears that a new paradigm on airway tightness in allergic inflammation is under development. during different time periods, separated by about a centennium, asthma has been characterized as a desquamative disease with denuded bronchial basement membranes. denudation has even been considered a hallmark of asthma and many research groups have lately employed denuded airways in in vitro contractility experiments to somehow mimic the asthmatic condition. it seems clear that in chronic inflammatory airway disease epithelial damage and shedding are increased. however, this may not necessarily mean that the airways will exhibit denuded basement membranes. a key question that may not have received sufficient attention concerns the epithelial repair-or restitutionprocess that would be set in motion as soon as shedding occurs. erjefalt et al. [8] [9] [10] recently have examined effects induced by and following from gentle epithelial cell removal in vivo in guinea-pig trachea. the employed in vivo model mimics epithelial shedding by not causing bleeding, or damage to the basement membrane. two important findings are the promptness and the high speed by which epithelial restitution starts and proceeds, respectively. the immediate in vivo responses to denudation are several-fold. the microcirculation responds by exuding bulk plasma and, with little delay, large numbers of neutrophils are extravasated (no bleeding occurs). thus, a plasma-derived fibrin-fibronectin gel rich in neutrophils soon covers the denuded basement membrane. this provisional cover is maintained and continuously supplied with plasma until a new tight epithelium has been established. the intact epithelial cells bordering the denuded area also respond immediately after loosing their neighbour cells. secretory and ciliated cells (and probably also basal cells) dedifferentiate, flatten and migrate over the membrane. the migration rate is particularly fast during the first minutes after denudation. the speed of migration, most likely aided by in vj'vo-specific factors, is so high (~3/;,m/min) that shedding, even of clusters of epithelial cells, would result in de-epithelialized basement membranes only for quite brief periods of time. hence, epithelial shedding even to the extent of denudation of limited areas may occur in vivo with little consequence to the mucosal barrier functions. defence and protection during the restitution process would be well catered for by the neutrophil-rich plasma-derived gel. this gel, with its content of plasma-derived migration-promoters such as fibronectin, fibrin and growth factors is obviously a suitable supramembranal milieu for high speed epithelial restitution. (in vitro studies dealing with epithelial repair demonstrate only relatively slow events.) complete denudation with loss of both columnar and basal cells may not be the most common kind of shedding. columnar cells may rather more easily be shed [55, 56] and thus leave a cobble-stone surface of basal cells behind. what happens to the basal cells when they loose their columnar neighbours? this question is currently being addressed by experimental approaches involving both animal and human airways [8] . it appears that the basal cells promptly undergo extensive flattening and that they establish extensive contact with each other. airway basal cells may thus be well suited to keep up the barrier function at shedding of ciliated and secretory cells. the new flat epithelium that is established after shedding or denudation consists of cells that have a larger apical surface than normal columnar epithelium. hence, a reduced length of paracellular stretches per unit mucosal area would be available for solute absorption. this structural change might explain the observations of reduced absorption in desquamative airway diseases. it is a separate matter that the limited sequelae to epithelial cell removal may also change our view on the role of epithelial shedding in respiratory defence. the intact airway epithelium has tight cell to cell contacts at the apical pole ofthe cylindrical cells. however, the tight mucosa still absorbs small molecules through paracellular routes. even proteins are absorbed but at slow rates. in exudative eosinophilic allergic disease the airway absorptivity may not be increased. it can even be decreased. this novel notion of mucosal tightness in inflammation is compatible with recent observations in two other areas of research: 'mechanisms of mucosal exudation of plasma' and 'mechanisms of restitution of epithelial lining cells after shedding'. when the intact airway mucosa is exposed to inflammatory agents including allergen, it responds with mucosal exudation of 'bulk' plasma. aided by a self-sustained hydraulic pressure, the large plasma proteins enter the airway lumen without increasing the airway absorption ability. this paracellular valve-like mechanism ofthe normal columnar epithelium makes luminal entry of plasma a significant flrst line defence mechanism [5] . sustained inflammatory processes cause epithelial damage, and epithelial shedding may be extensive in rhinitis and asthma. if only columnar cells are shed the remaining basal cells may promptly flatten out and tighten the barrier. if denudation occurs the basement membrane is immediately covered by a plasma-derived gel. the gel also provides a special milieu in which bordering ciliated, secretory, and basal cells dedifferentiate, flatten and migrate speedily. a new epithelial lining is established in such a milieu and at such high speed that epithelial shedding should probably also be considered a well functioning airway defence process. the new findings on basal cell responses at columnar cell losses and on reepithelialization in a plasma-derived gel in vivo after denudation may in part explain why increased absorption permeability has not been widely demonstrated in allergic and other inflammatory airway diseases. perhaps the clues to explain increased absorption tightness in asthma and rhinitis can also be found among mucosal exudation and repair mechanisms as they evolve under proper in vivo conditions. allergen, bradykinin, and capsaicin increase outward but not inward macromolecular permeability of guinea-pig tracheobronchial mucosa effects of histamine, ethanol, and a detergent on exudation and absorption across guinea pig airways mucosa in vivo absorption of ^'cr edta across the human nasal airway barriers in the presence of topical histamine plasma exudation as a first line respiratory mucosal defence subepithelial hydrostatic pressure may regulate plasma exudation across the mucosa asymmetrical effects of increases in hydrostatic pressure on macromolecular movement across the airway mucosa. a study in guinea-pig tracheal tube preparation basal cells promptly flatten out at detachment of the columnar epithelium in human and guinea-pig airways microcirculation-derived factors in airway epithelial repair in vivo in vivo restitution of airway epithelium ein neuer fall von asthma bronchial mit anatomischer untersuchung zur pathologischen anatomie des bronchialasthmas epithelial cell dysfunction in rhinitis epithelial pathology in asthma. a target for drug therapy primary sensitisation to inhalant allergens during infancy bronchial asthma as phenomenon of anaphylaxis permeability changes in obstructive airway disease a comparison of the immunologic responses of normal and atopic individuals to intranasally administered antigen immunologic responses of atopic and normal individuals to aerosolized dextran localization of antigen in experimental bronchoconstriction in guinea pigs effect of histamine and methacholine in guinea pig tracheal permeability to hrp effect of allergic bronchoconstriction on airways epithelial permeability to large polar solutes in the guinea pig nasal mucosal hyperpermeability to macromolecules in atopic rhinitis and extrinsic asthma the rate of absorption of ragweed pollen material from the nose comparative nasal absorption of allergens in atopic and nonatopic subjects ultrastruktur der allergischen nasenschleimhaut des menschen macromolecular permeability of the tight junction of the human nasal mucosa role of epithelium changes in permeability of dog tracheal epithelium in response to hydrostatic pressure stimulated eosinophils and proteinases augment the transepithelial flux of albumin in bovine bronchial mucosa oxidants altect permeability and repair of the cultured human tracheal epithelium effect of seasonal allergic rhinitis on airway mucosal absorption of chromiun-51-labelled edta the use of the nose to study the inflammatory response of the respiratory tract airway epithelium and microcirculation appearance of airway absorption and exudation tracers in guinea-pig tracheobronchial lymph nodes the 'nasal pool' device applies controlled concentrations of solutes on human nasal airway mucosa and samples its surface exudations/secretions plasma exudation in the airways: mechanisms and function the ultrastructural route of fluid transport in rabbit gall bladder volume flows across gallbladder epithelium induced by small hydrostatic and osmotic gradients mechanisms of glucagon-induced intestinal secretion epithelial pathways for luminal entry of bulk plasma plasma exudation, epithelial integrity and mucosal absorption ability in airways challenged with paf and h2o2 bronchotracheal response in human influenza effect of polymers and microspheres on the nasal absorption of insulin in rats persson eta\. nasal absorption of ^'cr-edta in smokers and control subjects airway mucosal exudation of plasma as a measure of subepithelial inflammation mechanisms of nasal hyper-reactivity erjefalt 1. toluene diisocyanate produces an increase in airway tone that outlasts the inflammatory exudation phase increased sensitivity to toluene diisocyanate (tdi) in airways previously exposed to low doses of tdi bronchial hyperresponsiveness, airway inflammation and occupational asthma induced by toluene diisocyanate microvascular exudative hyperresponsiveness in human coronavirusinduced common cold peptide absorption in human allergic airways exudative hyperresponsiveness to histamine in seasonal allergic rhinitis permeability of bronchial mucosa to "•'"in-dtpa in asthma and the effects of salmeterol the role of basal cells in adhesion of columnar epithelium to airway basement membrane the site of disruption of the bronchial epithelium in asthmatic and non-asthmatic subjects this work was supported by the swedish medical research council projects 8308,4499 and 2872, the medical faculty, university of lund; astra draco, lund; and the swedish association against asthma and allergy. we thank mai broman for the secretarial work. key: cord-018142-xt71w4nr authors: samy modeliar, s.; monge, m.; slama, m. title: thrombotic microangiopathy syndrome in the icu date: 2006 journal: yearbook of intensive care and emergency medicine doi: 10.1007/3-540-33396-7_20 sha: doc_id: 18142 cord_uid: xt71w4nr major studies designed to improve our understanding of the pathophysiology of tma have been conducted over recent years. this improved knowledge opens up new perspectives for more targeted treatment. however, until these innovative treatments become available, early diagnosis of these diseases is essential in order to rapidly initiate specific treatment, as the interval between diagnosis and initiation of plasma exchange is a decisive element in the prognosis of ttp. treatment must not be stopped too early or too rapidly and must take into account the various associated factors, especially the presence of infection. z pathophysiology the early phenomenon common to all forms of tma is damage to, or activation of, the vascular endothelium, responsible for local platelet aggregation, promoting the formation of platelet thrombi in the microcirculation. various factors are responsible for this endothelial cell activation: infections, drugs, cancers, stem cell transplantation, etc. [2] . role of von willebrand factor (vwf) and adamts-13 protein (a disintegrin and metalloprotease with thrombospondin type 1 motif). physiologically, vwf is a multimeric glycoprotein that triggers the formation of platelet clot, and transport of clotting factor viii. it is synthesized by megakaryocytes and endothelial cells, and then stored in endothelial and platelet weibel-palade bodies. ultra-large vwf multimers (ulvwf) are composed of several vwf monomers linked by disulfide bonds. vwf multimers have more intense hemostatic properties than monomers [2] . the adamts-13 protein is a metalloprotease that cleaves vwf multimers into monomers [3, 4] . ttp is associated with very low levels of adamts-13 protein activity 1 , resulting in elevated plasma levels of ulvwf [5] . in this context of metalloprotease deficiency, ulvwf are released in response to endothelial cell damage, and then accumulate and adopt an optimal procoagulant configuration, resistant to the shearing forces of the microcirculation. ulvwf bind to platelet gpiib and gpiib/iiia receptors, inducing excessive platelet aggregation. this mechanism is responsible for consumption thrombocytopenia and the formation of microthrombi decreasing the caliber of capillaries of the microcirculation, leading to tissue ischemia and erythrocyte fragmentation on thrombi (schistocytes) [2] . deficiency of the protease cleaving ulvwf in ttp is attributed either to acquisition of an igg inhibitory auto-antibody or to a mutation of the gene coding for the protease (familial cases) [6±10] . role of infections in the pathogenesis of ttp. tma is frequently associated with infection, whether or not there is another underlying disease [11] . in a retrospective intensive care study, 53% of cases of tma were associated with infection [1] . these infections have a major impact on the patient's outcome [11] . various microorganisms have been isolated during the acute phase of ttp, or during the days preceding ttp (table 1 [12±20] ). an underlying infection must be systematically sought in all cases of tma. the infectious agent involved in the pathogenesis of ttp induces a direct or indirect endothelial lesion (mediated by sepsis), as various mediators (interleukin [il]-1 and il-6, interferon gamma [ifn-c, tumor necrosis factor [tnf]-a) are released during the inflammatory reaction of sepsis and generate endothelial lesions. s. samy modeliar et al. damaged endothelial cells degranulate, releasing procoagulant substances (ulvwf, platelet-activating factor [paf]) into the plasma, express adhesion molecules and produce chemokines such as il-8. these phenomena promote adhesion and activation of neutrophils, which participate in endothelial lesions. activated endothelial cells also show decreased synthesis of prostaglandin i2 (pgi2), the most potent platelet aggregation inhibitor in the body. these mechanisms lead to a hypercoagulability state, which persists in individuals presenting predisposing factors. the pathophysiology of hus is characterized by intravascular coagulation specifically involving the renal microcirculation. two very different forms are distinguished: z post-diarrheal hus, usually endemic and mainly affecting young children (1±5 years), z hus occurring in the absence of diarrhea or atypical hus, observed in older children and adults. post-diarrheal hus [21] . this is the most frequent form of hus (90% of cases). the majority of cases are due to toxin-producing escherichia coli gastrointestinal infection. the serotype most frequently isolated is e. coli 0157:h7, but many other serotypes as well as many other bacteria have been incriminated ( table 2) . the e. coli toxin is called shiga-like-toxin (slt) due to its structural analogy with shigella dysenteriae toxin type 1. the pathophysiology of post-diarrheal hus starts with ingestion of food usually contaminated by a strain of toxin-producing e. coli. intestinal colonization by e. coli is responsible for liquid diarrhea. this colonization phase is accompanied by massive toxin production. this toxin is released into the intestine and is responsi-ble for direct microvascular and mucosal lesions, causing another episode of potentially hemorrhagic diarrhea. the toxin binds to specific receptors, is internalized and then enters the systemic circulation. e. coli bacteremia is not usually observed in post-diarrheal hus. the toxin is transported by neutrophils to target tissues, i.e., the kidney, where it binds to specific receptors situated on endothelial cells of the renal cortex and medulla and tubular epithelial cells. it is internalized in renal cells and induces inhibition of protein synthesis via ribosomal inactivation (depurinization), leading to apoptotic cell death. in addition to these lesions, the toxin also stimulates cytokine production. endothelial cell damage has several consequences: expression of tissue factor on the cell surface, activation of coagulation, release of paf into the circulation, and induction of overexpression of plasminogen activator inhibitor (pai)-1 on the endothelial cell surface. damaged endothelial cells also show decreased pgi2 production, and activate platelets and neutrophils predisposing to platelet aggregation on the endothelial cell surface. these phenomena lead to the formation of fibrin-rich microthrombi in renal capillaries, resulting in renal failure, formation of schistocytes and consumption thrombocytopenia. atypical hus [2, 22] . atypical hus is much rarer (7 to 10% of cases), occurring sporadically in adults and older children. it is characterized by the absence of gastrointestinal infection. its pathophysiology is poorly elucidated. various hypotheses have been proposed in adults (viral infections, bacterial infections, certain drugs, etc.) to be responsible for endothelial lesions and features of hus. a circulating factor present in the kidney has also been proposed. atypical hus in children has been reported to be associated with persistent consumption of the c3 fraction of complement via the alternative pathway [23] . decreased complement levels can be associated with factor h deficiency (factor h inhibits the alternative complement pathway). atypical hus with factor h deficiency usually corresponds to sporadic cases rather than familial cases and is rare in adults [24, 25] . there is no clearly established correlation between factor h deficiency and the development of atypical hus (factor h deficiency induces excessive c3 consumption, causing increased activation of neutrophils and excessive platelet aggregation). . this is a particular pathophysiological entity related to expression of the thomson friedenrich antigen on the surface of erythrocytes, endothelial cells, and glomeruli. this antigen, normally masked by sialic acid, is revealed by neuraminidase secreted by pneumococcus. this antigen is then recognized by circulating igm, leading to platelet aggregation and endothelial and glomerular lesions. z diagnosis tma disorders are rare in intensive care, with a prevalence of 0.35% of admissions according to a retrospective study conducted in adult intensive care units between 1998 and 2001 (64 patients) [11] . they represent 1 in 300 admissions, including cases of pre-eclampsia, stem cell transplantation, hellp syndrome and terminal cancer. early diagnosis is important (the prognosis is better when appropriate treatment is rapidly initiated). a diagnosis of tma must be considered in any case of sudden onset of poorly defined neurological symptoms and hematological abnormalities (especially thrombocytopenia and anemia). tma is observed in severely ill patients, often requiring admission to the icu. in a retrospective study of tma in 63 adult icu patients conducted in 14 french teaching hospitals, the mean simplified acute physiology score (saps) ii on admission was 45 ô 26 [11] . in another retrospective study, the mean saps ii score on admission was 37 ô 18 (30 patients) [1] . the criteria for admission to the icu are mainly life-threatening disease due to renal failure and/or neurological signs (convulsions, ischemia, coma). the other reasons for admission are hemorrhage, metabolic disorders (hepatocellular insufficiency), arrhythmias, myocardial infarction, etc [26] . clinical features of ttp [2, 27] the clinical presentation of ttp is marked by a sudden onset in an adult in previously good health. it has a female predominance (m/f ratio: 3/2) and generally occurs in the 4th decade. however, it can also occur in older or younger people. the estimated incidence is 4 per million inhabitants per year and has been constantly increasing over recent years. in 40% of cases, there is a prodromal phase resembling viral infection for several days before the acute episode (asthenia, arthralgia, myalgia, low back and abdominal pain). in 80% of cases, the triad suggestive of the diagnosis is observed immediately (mechanical hemolytic anemia, peripheral thrombocytopenia and neurological signs). in 40% of cases, two other cardinal signs are present (hyperthermia and renal failure). the diagnosis of ttp must always be considered in a patient with anemia and thrombocytopenia associated with organ failure. anemia is the most constant and earliest feature and consists of severe, regenerating (reticulocytes > 120,000 mm 3 ), normochromic, normocytic, hemolytic (elevated lactate dehydrogenase [ldh] and bilirubin, and very low haptoglobin), microangiopathic anemia. the blood smear reveals schistocytes, confirming the mechanical hemolysis (negative direct coombs test). the presence of schistocytes may be delayed and should be looked for on subsequent smears [28] . peripheral thrombocytopenia is marked (often less than 20,000/mm 3 ) and constant and reflects disseminated intravascular platelet hyperaggregability. the hemorrhagic manifestations related to this thrombocytopenia are varied in terms of site and severity. clotting tests are normal. fibrin and fibrinogen degradation products (fdp) may be observed in rare cases. neurological signs are present in only 60% of cases at the initial phase of the disease (and globally in 84 to 92% of cases during the course of the disease). they are characterized by their sudden onset and transient nature, and intermittent involvement of various territories over an interval of several hours. neurological signs comprise: obtundation, confusion, hemiparesis, dysarthria, aphasia, coma, disorders of consciousness, sensory deficit. deep tendon reflexes are often brisk. generalized convulsions are observed in 20% of cases and can evolve to status epilepticus. hyperthermia is present in 20% of cases at the initial phase, and then in 59 to 89% of cases during the course of the disease. it is usually only low-grade and reflects underlying hemolysis and cellular ischemia. renal failure occurs in one half of cases. it is usually moderate and, in the majority of cases, consists of macroscopic hematuria or slight proteinuria (less than 3 g/24 h). oligo-anuric renal failure is rare. other organ lesions may be observed, particularly myocardial infarction or myocarditis that may lead to acute cardiogenic shock and chronic heart failure. acute respiratory distress syndrome (ards) requiring mechanical ventilation, colonic ischemic, acute pancreatitis and sometimes ocular lesions may also be observed. sporadic ttp that resolves definitively is distinguished from recurrent forms with regular and frequent relapses, and intermittent ttp associated with irregular relapses [29] . hus comprises a combination of mechanical hemolytic anemia, peripheral thrombocytopenia and acute renal failure. the renal lesion is predominant in all forms and is classically associated with severe hypertension at the time of diagnosis. this hypertension is more severe and more frequent than during ttp, while neurological signs are less frequent than during ttp. anemia and thrombocytopenia have similar characteristics to those of ttp (see above). thrombocytopenia can be more severe than during ttp. clotting abnormalities may be observed: slight reduction of fibrinogen and clotting factors v and vii. elevation of fibrin degradation products, tissue plasminogen activator and pai reflect activation of homeostasis by tissue factor in the kidney. kidney needle biopsy is only performed when there is a doubt about the diagnosis or in the case of persistent renal failure [30] . epidemic (post-diarrheal) hus. post-diarrheal hus has a sudden onset in 90% of cases, presenting with diarrhea (bloody in 70% of cases and febrile in 50% of cases). it is essentially observed in children, but also in adults. the renal lesion appears one week after onset of the diarrhea (which has resolved at the time of diagnosis in 50% of cases). clinical manifestations are dominated by renal failure with severe proteinuria and hematuria (microscopic or macroscopic). fifty percent of children are anuric at the time of diagnosis. one half of patients with renal failure will require dialysis. severe hypertension is often associated with marked hyponatremia. central nervous system signs are present in 10 to 30% of cases, often accentuated by hyponatremia, consisting of irritability, drowsiness, convulsions or even coma. the microorganism responsible is often no longer present in the stools at the time of the diagnosis of hus, but the toxin can be isolated in two thirds of cases. the clinical and laboratory diagnosis of hus is usually easy to establish and kidney needle biopsy is rarely necessary. atypical hus. the clinical features are often less typical, and kidney needle biopsy is more often required to establish the diagnosis. acute renal failure is often anuric with moderate proteinuria (1 to 2 g/24 h), but proteinuria may also exceed 3 g/24h. microscopic hematuria is frequent and hypertension is present in 50% of cases at diagnosis. thrombocytopenia is present in only 50% of cases at diagnosis. there are few or no extra-renal signs (fever, neurological signs). a history of infection is frequently reported. adults sometimes present a context of auto-immune disease, drugs or pregnancy. the assessment of atypical hus must include assays of complement fractions (c3, c4, ch50), and the activity of the adamts-13 protein and the inhibitory protein of the alternative complement pathway (and a test for a mutation of the corresponding gene). the clinical features of hus and ttp tend to overlap and it can be difficult, on admission, to distinguish between these two diagnoses. the neurological signs are variable, ranging from simple confusion to coma with generalized convulsions and focal deficits, aphasia, diplopia, facial paralysis, etc. renal involvement is also heterogeneous, ranging from normal renal function to anuric acute renal failure. laboratory test results can confirm the diagnosis, but often only retrospectively. the prognosis of tma prior to plasma therapy in the icu was extremely poor. plasma therapy has considerably improved the prognosis of these usually fatal diseases (3-month mortality greater than 90% for acute forms of ttp without treatment) [27] . in a recent retrospective study of 64 patients admitted to the adult icu for tma, the mortality rate was 35% [11] and coppo et al. reported a mortality rate of 25% in intensive care patients with tma [1] . the mortality related to tma in intensive care is partly dependent on the treatment used. in recent studies [31±33], the survival rate of patients treated with plasma exchange is 78 to 90%. in the study by pene et al. [11] , the mortality at 90 days was 22% when plasma exchange was performed and 59% in the absence of plasma exchange. the study by rock et al. [33] confirmed the superiority of plasma exchange compared to fresh frozen plasma (ffp) infusion in terms of survival and response to treatment (78 versus 49%). the use of plasma exchange is positively correlated with survival. overall, tma syndromes with severe organ dysfunction requiring admission to the icu are associated with a high mortality. the main cause of death in these patients in the icu is multiple organ failure (mof). an infection associated with tma syndrome has a major impact on the outcome of these patients. the presence of a neurological deficit, possibly evaluated by the glasgow coma scale, is the main negative prognostic factor correlated with mortality [11, 32, 34, 35] . other prognostic factors have also been demonstrated: the saps ii score, need for vasoactive support, bilirubin level, ldh kinetics on the 3rd day of treatment [11] . predictive scores for ttp have been established, but their value has not yet been validated [35] . unlike other diseases usually observed in intensive care, the development of acute renal failure or the need for dialysis in a context of tma are not correlated with mortality [11, 33, 36] . according to lara et al. [32] , renal failure is correlated with a risk of relapse of tma, which has a poor prognosis, but the renal prognosis of ttp is generally good. the survival rates of post-diarrheal hus are excellent in response to symptomatic treatment alone, and admission to the icu is only rarely necessary. the mortality of post-diarrheal hus varies between 3 and 8% and is related to gastrointestinal and neurological lesions. the short-term renal prognosis is good (improvement of renal function over several days to several weeks). however, one quarter to one third of children still present renal sequelae 10 years later (proteinuria and/or renal failure) with end-stage chronic renal failure in 3 to 4% of cases. atypical hus in children has a poor prognosis, frequently resulting in end-stage chronic renal failure and a high post-transplantation recurrence rate. atypical hus in adults is a serious disease in which the prognosis depends on the etiology of hus. the concomitant presence of advanced cancer, hiv infection, mitomycin c therapy and post-partum etiology carries a very poor prognosis. the intensive care mortality rate of atypical hus is 10 to 20%. twenty-five to 50% of these patients will require chronic dialysis. only 50% of survivors are completely cured without sequelae. in patients with underlying nephropathy, chronic renal failure persists after the acute episode in 70% of cases. long-term recurrences are possible, especially when the cause of hus persists, but are less frequent than for ttp. z management tma always requires emergency treatment. admission to intensive care, in addition to the usual criteria of organ failure, must be proposed in all patients with severe thrombocytopenia (less than 50,000/mm 3 ), due to the high frequency of organ dysfunction at the acute phase of the disease. symptomatic treatment is required in all forms of tma. tma disorders always have an unpredictable course. all available measures of`aggressive intensive care' must, therefore, be proposed, even in the case of severe neurological signs. patients with respiratory failure require mechanical ventilation. this corresponds to 59% of all patients admitted to the icu for tma [11] . when possible, non-invasive ventilation should be preferred to invasive ventilation due to the hemorrhagic and infectious risks associated with invasive ventilation. patients with severe renal failure require dialysis (47% of tma patients in intensive care) and 32% of patients require vasopressor support. hypertension must also be treated, preferably by angiotensin converting enzyme (ace) inhibitors. the target blood pressure is 120/80 mmhg. folate supplementation is systematically prescribed (intense folate-consuming bone marrow regeneration). anemia must be corrected by packed cell transfusion to achieve hemoglobin concentrations greater than 8 g/dl. platelet transfusions should be avoided, except in the case of uncontrolled bleeding, as they may increase the thrombotic process in the microcirculation. anticonvulsants should be prescribed to patients with a history of epilepsy. any triggering or associated factors must be treated. in particular, antibiotics should be initiated whenever an infection is suspected (except for cases of post-diarrheal hus, in which antibiotics can worsen the hus [37] ). persistence of an occult bacterial infection can lead to persistence of ttp, making it refractory to conventional treatment. indication. this treatment is not indicated in post-diarrheal hus, as plasma therapy does not modify survival (which is excellent in response to symptomatic treatment alone). plasma therapy must be urgently initiated in patients with ttp, as its efficacy in terms of survival has been demonstrated. plasma therapy is used in adults with hus by analogy with the management with ttp, although no studies are in favor of either plasma exchange or ffp transfusion. it is essential to control underlying predisposing factors. fresh frozen plasma transfusion or plasma exchange? recent studies have demonstrated the superiority of plasma exchange over ffp transfusion in terms of survival and response to treatment of ttp [36] . the use of plasma exchange is independently correlated with survival. the efficacy of plasma exchange depends on the volume of plasma administered to the patient (plasma exchange is able to administer three times more plasma than ffp transfusion alone [11] ). two pathophysiological mechanisms can explain the efficacy of plasma exchange: plasma exchange removes a`pathogenic' component from the patient's plasma (ulvwf or auto-antibodies) and compensates for the deficient protease (by providing large quantities of plasma). in cases of ttp not associated with an inhibitor, ffp transfusions may be sufficient. when plasma exchange cannot be started immediately (for technical reasons, or while waiting for transfer to a specialized center), large-volume ffp transfusion (20 to 30 ml/kg/day) can be started prior to plasma exchange, but it is associated with a risk of fluid overload, protein-overload proteinuria, hyperproteinemia, and hyperviscosity syndrome. the raised oncotic pressure can also accentuate renal insufficiency. the plasma exchange technique is based on the daily exchange of one to two plasma masses fully compensated by ffp. a plasma exchange session lasts about 3 hours for 50 to 80 ml/kg of ffp. it is performed via a central venous catheter, rarely placed in a subclavian vein due to the risk of bleeding, and requires anticoagulation at effective doses during the session. a systematic calcium supplement is started during plasma exchange (risk of hypocalcemia due to the toxicity of citrate). plasma exchange is time-consuming, expensive and invasive and carries a risk of immunological pulmonary edema (almost eliminated by solvent/detergenttreated plasma). the duration of treatment is variable. plasma exchange is continued daily until restoration of a normal platelet count (> 150,000/mm 3 ) for at least 48 hours with a reduction in ldh levels and reticulocyte counts. the frequency of plasma exchange must be progressively decreased. daily plasma exchange must be reintroduced at the slightest sign of relapse. in the future, monitoring of adamts-13 protein inhibitor levels could guide the frequency of plasma exchange. during relapses (30% of cases of ttp), the initial treatment in the acute phase is the same as for the first episode. repeated relapses may represent an indication for splenectomy, during a period of remission. refractory ttp, defined by no improvement of the platelet count on day 5 of treatment, requires twice-daily plasma exchange and adjuvant therapies (vincristine, polyvalent immunoglobulins, cyclophosphamide: see below) [38, 39] . high-dose corticosteroid therapy is effective in 56% of purely hematological forms of ttp [31] , although its efficacy has not been clearly demonstrated by randomized trials. in the absence of a contraindication (active infection), and despite a low level of evidence, methylprednisolone (1 mg/kg/day for 3 weeks) can be administered immediately following plasma exchange. the therapeutic value of platelet aggregation inhibitors, vincristine, high-dose immunoglobulins, splenectomy, immunosuppressives, unfractionated heparin, fibri-nolytics, prostacyclin, and vitamin e in the intensive care management of tma has not been demonstrated. staphylococcal protein a columns could be effective in ttp, especially in a context of cancer, but they have not been evaluated in patients with an inhibitor [40] . a possible treatment for the future would be purified or recombinant protease infusion, which could replace plasma therapy. z other tma syndromes pregnancy-associated tma various types of tma can occur during pregnancy and the post-partum period: ttp, hus, hellp syndrome. many complications have been described including acute necrotic pancreatitis, myocardial infarction, gastrointestinal ischemia, etc. the treatment of ttp during pregnancy is based on plasma exchange. pregnancy does not modify the response to treatment, but the consequences of plasma exchange on the fetus have not been evaluated. there is a risk of relapse during subsequent pregnancies. ante-partum hus requires termination of pregnancy, but has a fairly good prognosis. post-partum hus can occur up to 3 months after delivery and has a poorer prognosis. death may be due to cerebral ischemic or hemorrhagic lesions or cardiac sudden death. some cases of post-partum hus are related to factor h deficiency (heterozygous mutation). the hellp syndrome (hepatic form of tma) is a tma disorder specific to pregnancy. it differs from hus and ttp by the presence of dic and liver impairment. treatment consists of fetal extraction. plasma therapy has been proposed (3 to 5 post-partum sessions appear to be effective). there is a risk of recurrence during subsequent pregnancies. these patients should preferably be treated in a specialized center. in this context, tma is triggered by various predisposing factors including total body irradiation, infections (immunodepressed patients), drugs (tacrolimus, cyclosporine a), and acute graft versus host disease. these predisposing factors induce disseminated endothelial damage. the adamts-13 protein level is normal and the response to treatment is disappointing. plasma exchange improves the prognosis, which nevertheless remains poor. management of predisposing factors is an essential aspect of treatment. tma is essentially associated with secretory adenocarcinomas, such as breast or stomach cancers, or more rarely lung, colon or prostate cancers. stomach cancers represent more than 50% of all reported cases of cancer-associated tma. in most cases, the cancer has already been diagnosed and treated, but ttp can also be the first sign of cancer. the pathophysiology of cancer-associated tma has been poorly elucidated and is associated with variable adamts-13 protein levels. the prognosis is also variable and depends on the underlying cancer. drug-or toxin-associated tma many drugs have been incriminated or suspected including ticlopidine, clopidogrel, cyclosporine a, tacrolimus, interferon alpha, oral contraceptives, quinine, cisplatin, mitomycin c, bleomycin, arsenic, penicillamine d. ticlopidine and clopidogrel are associated with severe adamts-13 protein deficiency with the presence of plasma inhibitors. plasma exchange therapy achieves complete remission in the majority of these patients [41, 42] . tma associated with mitomycin c is dose-dependent and is observed after a total dose of more than 60 mg (2 to 10% of treated patients). the tma disorders often appear several months after stopping mitomycin c and are characterized by hypertension and pulmonary edema [43] . neurological disorders and hyperthermia are rarely present. the prognosis is very serious despite discontinuation of mitomycin and treatment by plasma exchange (mortality of 60 to 70%). hiv-associated tma [12] these forms of tma generally occur at an advanced stage of hiv infection and, therefore, have a very poor prognosis. various mechanisms can be responsible: z development of anti-adamts-13 auto-antibodies responsible for ttp that responds favorably to treatment z a multifactorial origin (opportunistic cmv infection [44] ), drugs (valaciclovir, etc.) with a more variable response to treatment and a poorer prognosis. treatment of this type of tma syndrome must be associated with antiretroviral therapy. other diseases have also been associated with tma disorders, including catastrophic antiphospholipid syndrome, veno-occlusive disease, dic, type 2 heparininduced thrombocytopenia, giant hemangioma, hemangio-endothelioma, and malignant hypertension. these various syndromes must be eliminated before initiating plasma therapy. z conclusion major studies designed to improve our understanding of the pathophysiology of tma have been conducted over recent years. this improved knowledge opens up new perspectives for more targeted treatment. however, until these innovative treatments become available, early diagnosis of these diseases is essential in order to rapidly initiate specific treatment, as the interval between diagnosis and initiation of plasma exchange is a decisive element in the prognosis of ttp. treatment must not be stopped too early or too rapidly and must take into account the various associated factors, especially the presence of infection. infectious diseases as a trigger in thrombotic microangiopathies in intensive care unit (icu) patients? thrombotic thrombocytopenic purpura and other thrombotic microangiopathy syndromes purification of human von willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family partial amino acid sequence of purified von willebrand factor-cleaving protease thrombotic microangiopathies willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome mutations in a member of the adamts gene family cause thrombotic thrombocytopenic purpura willebrand factor cleaving protease (adamts13) is deficient in recurrent and familial thrombotic thrombocytopenic purpura and hemolytic uremic syndrome antibodies to von willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura specific von willebrand factor-cleaving protease in thrombotic microangiopathies: a study of 111 cases outcome of severe adult thrombotic microangiopathies in the intensive care unit human immunodeficiency virus infection and thrombotic microangiopathy thrombotic microangiopathy in association with cytomegalovirus infection in a renal transplant patient: a new treatment strategy cryptococcal meningitis following a thrombotic microangiopathy in an unrelated donor bone marrow transplant recipient hemolytic-uremic syndrome associated with pneumococcal sepsis thrombotic thrombocytopenic purpura complicating legionnaires' disease thrombotic thrombocytopenic purpura and herpes zoster infection thrombotic thrombocytopenic purpura (ttp) associated with a borrelia burgdorferi infection thrombotic thrombocytopenic purpura associated with bacteroides bacteremia thrombotic thrombocytopenic purpura associated with primary tuberculosis hemolytic uremic syndrome insuffisances rnales aigus d'origine glomrulaire et vasculaire recurrent haemolytic uraemic syndrome and acquired hypomorphic variant of the third component of complement heterozygous and homozygous factor h deficiencies associated with hemolytic uremic syndrome or membranoproliferative glomerulonephritis: report and genetic analysis of 16 cases plasma exchange for treatment of thrombotic thrombocytopenic purpura in critically ill patients purpura thrombotique thrombocytopnique the hemolytic uremic syndrome recent advances in thrombotic thrombocytopenic purpura prothrombotic coagulation abnormalities preceding the hemolytic-uremic syndrome improved survival in thrombotic thrombocytopenic purpura-hemolytic uremic syndrome. clinical experience in 108 patients improved survival with plasma exchange in patients with thrombotic thrombocytopenic purpura-hemolytic uremic syndrome thrombotic thrombocytopenic purpura: outcome in 24 patients with renal impairment treated with plasma exchange thrombotic thrombocytopenic purpura/ hemolytic uremic syndrome: a multivariate analysis of factors predicting the response to plasma exchange high incidence of relapses in thrombotic thrombocytopenic purpura. clinical study of 38 patients comparison of plasma exchange with plasma infusion in the treatment of thrombotic thrombocytopenic purpura. canadian apheresis study group the risk of the hemolytic-uremic syndrome after antibiotic treatment of escherichia coli o157:h7 infections thrombotic thrombocytopenic purpura: early and late responders rituximab for chronic recurring thrombotic thrombocytopenic purpura: a case report and review of the literature treatment of plasma refractory thrombotic thrombocytopenic purpura with protein a immunoabsorption thrombotic thrombocytopenic purpura associated with clopidogrel thrombotic thrombocytopenic purpura associated with ticlopidine. a review of 60 cases hemostatic abnormalities and increased vascular endothelial cell markers in patients with red cell fragmentation syndrome induced by mitomycin c thrombotic microangiopathy and cytomegalovirus disease in patients infected with human immunodeficiency virus key: cord-336177-p7b7yw28 authors: selvi, valeria title: convalescent plasma: a challenging tool to treat covid-19 patients—a lesson from the past and new perspectives date: 2020-09-22 journal: biomed res int doi: 10.1155/2020/2606058 sha: doc_id: 336177 cord_uid: p7b7yw28 on march 11(th), 2020, the world health organization declared covid-19 infection as a pandemic. since it is a novel virus, there are basically no proven drugs or therapies; although many laboratories in different countries are working to develop a vaccine, it will take time to make it available. passive immunization is the therapy born from the intuition of behring and kisato in the late 19(th) century. it was widely used for the treatment of bacterial infections until the discovery of antibiotics, as well as during the viral pandemics of the 20(th) century and of the beginning of the 21(st); it still has clinical applications (e.g., tetanus prevention). this paper summarizes the basic principles of passive immunization, with particular reference to convalescent plasma. the literature concerning its use during past epidemics and the results of the first clinical studies concerning its use during the current pandemic are discussed too. a large section is dedicated to the analysis of the possible, although rare, side effects. recently, in 2017, the who blood regulators network (brn) published a position paper, recommending convalescent plasma as the first-choice treatment to be tested in the absence of authorized drugs; however, this strategy has not been followed. in the current epidemic, the principle of passive immunization through convalescent plasma has been applied in several circumstances and particularly in patients with serious complications. the first reported results are encouraging and confirm the effectiveness of plasma therapy and its safety. also, the fda has proposed plasma treatment in order to face the increasingly complex situation and manage patients with serious or immediately life-threatening covid-19 disease. several studies and clinical programs are still ongoing. on march 11 th , 2020, the world health organization (who) declared covid-19 infection as a pandemic [1] . the virus causing covid-19 infection is a coronavirus called sars-cov-2; it began to scare the world since the first days of 2020 during its initial outbreak in china, because of the characteristics of contagion (high rate of contagiousness associated with high lethality) [2] . since it is a novel virus, there are basically no proven drugs or therapies. in hospitals all over the world, there are many ongoing clinical studies. many attempts have been made to treat seriously sick patients, using off-label drugs already known; nevertheless, to date, there is no effective targeted antiviral therapy. in most cases, drug administration has been authorized for a compassionate purpose [3] . in fact, who management of covid-19 has been mainly focused on infection prevention, case detection, and monitoring; supportive care and nonspecific anti-sars-cov-2 treatment have been recommended [4] . extensive vaccination is the only strategy to prevent pandemic transmission of sars-cov-2. major efforts are currently being made by many laboratories in several countries to develop a vaccine; however, it will still take time before the vaccine is widely available to the population [5] . encouraging news about passive immunization arrived from china at the end of february, and some authors reported them in their scientific publications. cai et al. cited two official sources (national health and health commission, health bureau of the logistics support department of the central military commission; chinese society of blood transfusion) reporting significant improvements in patients affected by covid-19 and treated with plasma donated by recovered patients [5] . anecdotal experiences are reported by keith et al. [6] and from cunningham et al. [7] ; in particular, the latter group reported that biotec group co. announced that 10 seriously ill patients, treated with immunoglobulin therapy, demonstrated improved oxygenation and reduced inflammation and viral load [7] . in addition, casadevall and pirofski, referring to the news from the xinhua news source, reported that convalescent serum was used for the therapy of 245 patients with covid-19 in china during the first outbreak. although few details are available and published studies involve a small number of patients, the authors concluded that convalescent serum is safe and reduces viral load [8] . additionally, convalescent plasma could potentially be used to prevent disease in high-risk cases (vulnerable individuals with underlying medical condition, health care providers, and individuals exposed to confirmed cases of covid-19 [8] . the literature about convalescent plasma is rapidly growing. the uniqueness of this work is that it presents all the main aspects about convalescent plasma in a single body. the already published review articles often focused on single aspects of convalescent plasma, and to the best of the author's knowledge, there are no available works covering all the main topics concerning convalescent plasma use. the present manuscript is intended to be a complete and update guide for doctors and institutions. a systematic search was conducted in major electronic databases (pubmed and medline) and google scholar; the applied query was "plasma or convalescent plasma" and "covid-19 or sars-cov-2". virus neutralization by antibodies is the principle behind the functioning of plasma of patients recovered from sars-cov-2; high-titer-specific antibodies bind to sars-cov-2 neutralizing the viral particles, blocking access to cells, and activating potent effector mechanisms, such as complement activation and phagocytosis [7] . there are several ways to achieve passive immunization. antibodies can be delivered to the recipient by (i) human whole blood, (ii) human or animal plasma or serum, (iii) pooled human immunoglobulin for intravenous (ivig) or intramuscular (ig) use, (iv) high-titer human immunoglobulin for intravenous or intramuscular use from immunized or convalescing donors, and (v) monoclonal antibodies (mab) [9, 10] . transfusion of whole blood to provide convalescent plasma should be avoided unless its use is clinically indicated; collection of convalescent plasma should be performed only by apheresis to avoid unnecessary red cell loss in the donor [11] . plasma administration is the preferred method to provide passive immunity in pandemic scenarios at least in the immediate term; usually, immunoglobulins are prepared by fractionating large pools of human plasma collected from approximately 10,000-40,000 donors [12] . moreover, plasma administration represents a rapid and effective therapy, has lower costs than other methods [13] , and presents a broader spectrum response. studies suggest that it not only neutralizes the pathogen but also provides passive immunomodulatory mediators allowing the recipient to control the excessive inflammatory cascade induced by the infectious agent [14] . animal plasma collection should be avoided if possible as it can cause side effects collectively called "serum sickness" [15] . convalescent plasma is not a novel therapy; it is a therapy widely used in the past, both for bacterial and viral pathologies. behering and kisato were the first in 1890 to provide the basis of passive immunization, then known as serum therapy. despite limited knowledge on the structural and functional complexity of antibodies, they demonstrated that not previously immunized animals can be protected from sublethal doses of diphtheria and tetanus toxin with serum therapy. the discovery was so important that in 1901, behring earned his noble prize for it. given the early success in the 1900s, passive immunization was rapidly expanded; it was used to treat several bacterial infections including corynebacterium diphtheriae, streptococcus pneumoniae, streptococcus pyogenes, clostridium tetani, haemophilus influenzae, and neisseria meningitidis; type-specific antipneumococcal serum was used as the first-line treatment for lobar pneumonia [15] . during the first half of the 20 th century, serum therapies were successfully used to treat patients affected by many infectious diseases (anthrax, plague, scarlet fever, measles, tularemia, diphtheria, dysentery, meningococcal meningitis, rabies, and pneumococcal pneumonia) [16] . however, serum therapy for bacterial diseases suddenly stopped after the discovery of antibiotics. moreover, the tools for the correct selection of plasma were still missing; the risk of serum disease was very high in those years, as plasma was frequently prepared from the blood of hyperimmunized animals [15] . regarding viral diseases, studies about convalescent plasma use to fight pandemics of the last century are available; the reported results were positive [8] . in the early 20 th century, convalescent serum was used to fight outbreaks of viral diseases such as poliomyelitis, measles, mumps, and spanish influenza [8] . a meta-analysis by luke and colleagues reported eight studies involving 1,703 patients with 1918 influenza pneumonia from 1918 to 1925. patients were often selected among the most serious ones and received an infusion of influenza convalescent human blood products; the outcome of the treated patients was compared to that of the untreated influenza pneumonia controls. the study showed a pooled absolute reduction of 21% in the mortality rate compared to controls. unfortunately, the included studies were few and with methodologic limitations (no study was a blinded, randomized, or placebo-controlled trial; moreover, convalescent 2 biomed research international sera were developed and used in many cases without measuring antibody titers or without knowledge about viral serotypes); anyway, this treatment received consensus at the time, and it was applied in several countries [4, 8, 17] . in the modern era, the treatment of argentine hemorrhagic fever (junin virus) with convalescent immune plasma was applied as part of a nationally organized response; patients treated with immune plasma had a much lower mortality than those given normal plasma [16, 18] . convalescent plasma or immunoglobulins were administered as a last chance to reduce the mortality rate of patients with sars; several studies showed a shorter hospital stay and lower mortality in patients treated with convalescent plasma compared to those not treated with convalescent plasma. the largest study involved the treatment of 80 patients showing clinical deterioration despite treatment with methylprednisolone. earlier plasma administration was more likely to be effective: patients treated before the 14 th day had better prognosis compared to those treated later. in addition, patients who were pcr positive and seronegative for coronavirus at the time of therapy had improved prognosis. no immediate adverse reactions were observed [4, 8, 19] . positive evidence was reported for the treatment of influenza a (h5n1) too [20] . regarding the pandemic 2009 influenza a h1n1, the results from the prospective cohort study by hung and colleagues showed that plasma treatment reduced mortality (the patients involved in the study were seriously ill and required intensive care); no adverse events were observed [4, 8, 20] . a second trial by hung and colleagues was conducted on 35 patients affected by severe influenza a h1n1 during 2010 and 2011, using immunoglobulins fractionated from plasma of patients recovered from the previous 2009 influenza a h1n1. treated patients showed a lower viral load and reduced mortality rate within 5 days of symptom onset [4, 8, 21] . a meta-analysis by mair-jenkins and colleagues, including 32 studies of sars coronavirus and severe influenza, reported that convalescent plasma reduced mortality and it was safe (no relevant adverse events or complications after treatment were reported). the reduction of mortality was higher when convalescent plasma was administered earlier after symptom onset [22] . regarding ebola disease, the use of convalescent plasma was recommended by the who in 2014 as an empirical treatment during the outbreaks [8] . the first use of convalescent plasma for ebola dates back to previous times. the study of mupapa et al. involved eight patients during an outbreak in 1995; among these, seven survived [23] . the ebola-tx clinical trial tested the efficacy of convalescent plasma as a treatment for ebola in guinea: the trial confirmed convalescent plasma safety, but unfortunately, the efficacy was not proven. no association with the dose of neutralizing antibodies was apparently found, even if the levels of neutralizing antibodies were low in many plasma donations. the authors concluded that further studies were needed to assess the effectiveness of antibody doses higher than those used in their study [24] . sahr et al. used convalescent serum in sierra leone for ebola treatment: their study revealed a significantly lower fatality rate for patients treated with convalescent whole blood with respect to those receiving standard treatments [13] . a protocol for convalescent plasma in the treatment of middle east respiratory syndrome (mers) caused by a coronavirus was established in 2015. three patients with mers in south korea were treated with convalescent serum, but only two showed neutralizing activity. the authors concluded that high antibody titer (≥1 : 80) should be needed to achieve good neutralization activity [4, 8, 25] . keller and stiehm listed all the pathologies for which passive immunization has been or is currently being used. for each pathology, they specified when passive immunization is to be used for prevention versus treatment and if the efficacy has been demonstrated (they also pointed out when there is no recommendation to use the passive immunization tool, even in the case of demonstrated efficacy). more than 30 infectious pathologies were analysed. the efficacy of passive immunization in the prevention of infectious diseases has been proven for tetanus, clostridium botulinum, hepatitis a, hepatitis b, rsv (respiratory syncytial virus), cmv (cytomegalovirus), vzv (varicella zoster virus), rabies, measles, and vaccinia. in addition, passive immunization has been proven but not recommended for the treatment of respiratory infections (streptococcus, streptococcus pneumoniae, neisseria meningitidis, and haemophilus influenzae) or for enterovirus infection. the efficacy of passive immunization in the treatment of infectious disease has been proven for diphtheria, tetanus, clostridium botulinum, and vaccinia and has been proven but not recommended for respiratory infections (streptococcus, streptococcus pneumoniae, neisseria meningitidis, and haemophilus influenzae), parvovirus, and enterovirus [9] . as already discussed, previous studies on convalescent plasma in pandemic scenarios have shown that plasma is a safe treatment [4, 8, 13, [17] [18] [19] [20] [21] [22] [23] [24] [25] . anyway, side effects are possible for any medication; maclennan and barbara analysed the possible side effects of generic plasma administration (not only convalescent plasma) in a recipient. several factors can lead to adverse events (donor-related factors, which testing is performed on plasma, any treatment or modification to which it has been subjected, interaction between donor factors, and the patient's immune system). possible adverse reactions can be classified into three groups: immune reactions (anaphylactic/anaphylactoid reactions, mild allergic reactions, haemolysis, and transfusionrelated acute lung injury), physicochemical reactions (fluid overload, citrate toxicity, and chemicals), and infectious risks. anaphylactic reactions are uncommon, but severe and potentially life-threatening (in 2003, the incidence in the uk was approximately 0.002%). ige mediates anaphylactic reactions; the term "anaphylactoid" describes a similar reaction not mediated by ige. less severe allergic reactions are much more common and usually characterized by cutaneous symptoms, ranging from mild pruritus to urticaria and flushing. haemolysis can occur following transfusion of plasma containing high-titer anti-a or anti-b haemolysins to an a or b recipient. it could be very serious, and deaths have also been reported. to avoid this reaction, plasma should always be abo compatible; if not possible, the plasma should be tested for haemolysins and found negative for high titer of them. concomitant transfer of antibodies against other red cell antigens might occasionally cause haemolysis in the recipient. thus, donor screening procedures to detect clinically significant antibodies are essential to minimize this risk [26] . trali is an acute respiratory reaction, indistinguishable from the adult respiratory distress syndrome (ards), occurring in association with transfusion of blood components; the incidence was reported variously, ranging from 1 in 5,000 to 1 in 50,000. it is caused by the presence of antibodies against leucocyte antigens (hla antigens seem the most frequent [11] ) in donor plasma [26] . to avoid this risk, preference should be given to the use of plasma from male donors or from females who have never been pregnant, including abortions. this measure lowers the possibility to find antibodies against hla or granulocyte antigens causing trali in the donor plasma [11] . moreover, it was reported that the presence of certain antibodies may cause immune enhancement of pathogenicity, termed ade (antibody-dependent enhancement), for several viral diseases, such as dengue virus and sars [27] . physiochemical reactions can sometimes be severe, but usually not life-threatening: (i) fluid overload is one of the most common complications of transfusion and can lead to pulmonary oedema (ii) citrate toxicity depends on the action of citrate in binding calcium and therefore in reducing the availability of ionised calcium for normal neuromuscular function; it is not frequent because citrate is rapidly metabolised by the liver (iii) some units of plasma might contain chemicals (e.g., drugs) derived from the donor to which the recipient might react modern technologies allow to minimize infectious risk. firstly, bacterial transmission is not a significant risk factor as the plasma is frozen within hours of collection and processing. secondly, an accurate selection of donors and pathogen reduction processes can be applied to minimize viral infection risk (for single-unit components, methylene blue is used; for plasma pools, solvent detergent is used) [26] . specific side effects are identified for single plasma components; immunoglobulins have been associated with thrombotic events, renal toxicity, and aseptic meningitis [26] . tamburello and marando reported that treatment with human immunoglobulin during the sars-cov-2 pandemic was associated with a significantly increased risk of same-day thrombotic events (from 0.04 to 14.9%) [3] . however, the estimated risk of serious adverse events is less than 5% [26] . a recent work by joyner and colleagues explores the safeness of the use of convalescent plasma in 20,000 critically ill covid-19 patients. the cohort studied is very huge; thus, the reported results should be considered particularly reliable. serious adverse events within 4 hours of completion of covid-19 plasma transfusion were 146 (less than 1% of all transfusions). 50 events were judged surely unrelated to plasma transfusion. among the other events, there were 83 nonmortality events reported (37 reports of transfusionassociated circulatory overload, 20 reports of transfusion acute lung injury, and 26 reports of severe allergic transfusion reaction). 13 mortality events happened; they were judged only as possibly, not definitely, related to the transfusion of covid-19 convalescent plasma. notably, the vast majority of other serious adverse events, which happened within seven days of completion of the convalescent plasma transfusion, were judged to be unrelated to the plasma transfusion [28] . recently, in 2017, the who blood regulators network (brn) published a position paper, recommending the need for healthcare systems to prepare adequate infrastructures to deal with the emergence of any pandemic caused by new emerging viruses; in that paper, the brn suggested plasma from recovered patients as the first-choice treatment to be tested. based on the evidence from past experience in passive immunization, the brn explained that there was a considerable possibility that the application of whole blood (as well as plasma, serum, or immunoglobulin concentrates) from convalescent persons could be effective in the treatment/prevention of infectious disease. thus, in the absence of effective vaccines and antiviral therapies for the emerging pathogen, an organized program to collect convalescent plasma or serum from disease survivors could provide a potentially valuable empirical intervention, while data on the effectiveness and safety of its use are obtained through orderly scientific studies [16] . in fact, any blood derivative should be considered a drug, and if administered for different indications from the authorized ones, it must be tested for the specific new application [29] . epstein [31] . unfortunately, data from case reports and case series are observational, and they are not sufficient for a definitive validation of the treatment. some controlled trials are already available too; they confirm the outcomes of the first case series. a randomized control trial out of wuhan was the first to be published: 103 patients with severe or life-threatening covid-19 (52 in the convalescent plasma-treating group and 51 in the control group) were enrolled, but unfortunately, the study had an early termination due to low patient enrollment as the regional outbreak waned. contrary to expectations, the study failed to detect a statistically significant difference in the evaluated outcomes (time to clinical improvement, 28-day mortality, and time from randomization to discharge). however, convalescent plasma was demonstrated to be associated with antiviral activity in patients with covid-19 (convalescent plasma treatment was associated with higher rates of negative sars-cov-2 viral pcr results from nasopharyngeal swabs at 24, 48, and 72 hours); a statistically significant improvement was noted for the convalescent plasma treatment group compared to controls in the subgroup of patients without life-threatening covid-19 (91% improvement in the plasma group compared to 68% in the control arm). the median between the onset of symptoms and the beginning of the treatment was 30 days. this time window could have affected the study to detect a clinically important benefit of the convalescent plasma therapy, in addition to the early termination of the study and to the type of patient conditions (only severe or lifethreatening disease) [32] . the . they demonstrated that convalescent plasma is associated with reducing ventilatory requirements in patients with both severe and life-threatening diseases [33] . these results are consistent also with a recent cohort study by liu and colleagues. in this study, 39 patients were treated with convalescent plasma and were compared to 156 control patients. the authors reported a lower mortality rate among patients with severe or worse disease who received convalescent plasma and significantly better outcomes among patients transfused prior to mechanical ventilation [34] . salazar et al. enrolled 387 patients (136 transfused patients and 251 nontransfused control covid-19 patients); they found that patients transfused within 72 h of hospital admission had decreased mortality within 28 days, whereas patients transfused after 72 h of hospital admission did not. these data demonstrate that early convalescent plasma transfusion after hospital admission reduces mortality within 28 days posttransfusion [35] . two other articles deserve to be mentioned, the one of duan et al. and the other of joyner et al. duan et al. compared 10 severely ill patients treated with convalescent plasma to a historical control group of 10 severely ill patients not treated with convalescent plasma. the covid-19-transfused patients' group showed better clinical outcomes than the historical control group. all enrolled severe covid-19 patients had improvement of clinical symptoms and showed different degrees of absorption of the pulmonary lesions after convalescent plasma transfusion. the authors showed amelioration of routine laboratory criteria and pulmonary function (lymphocytopenia, an important index for prognosis in covid-19, tended to be improved after convalescent plasma transfusion). increase of neutralizing antibody titers was demonstrated [36] . joyner et al. studied the effects of convalescent plasma use in a very huge cohort of 20,000 critically ill hospitalized patients. the aim of the paper differs from the ones previously mentioned: the study was designed to demonstrate the safety of convalescent plasma. anyway, the seven-day mortality rate in this extremely high-risk cohort of patients was 8.6% only. the authors anticipated the intent to create a control comparator group using patients hospitalized with covid-19 during the same period; they will discuss potential convalescent plasma efficacy in a future publication. given the large number of observations, it is expected that the results of this study will have significant importance in evaluating the efficacy of the treatment and its reliability [28] . moreover, there are several ongoing randomized controlled trials on the role of convalescent plasma to treat covid-19 (zheng et al. estimated that the main underway trials are 48 in the world). also in this case, a positive confirmation of the results in terms of the efficacy of the treatment for covid-19 is strongly expected [37] . preparation requirements for convalescent plasma follow the standard operating procedures for plasma collection and all applicable regulations. thus, health system requirements are the same for routine plasma collection procedures via plasmapheresis. during plasma donation procedure, the blood cells and plasma are removed from the body and separated by a plasmapheresis machine; then, the blood cells are returned to the donor while plasma is collected. plasma products are stored as fresh-frozen plasma, until usage. recently, approved serological assays are necessary to detect sars-cov-2 (rt-pcr test) in serum and virologic assays [35] . regarding plasma treatment in the context of the current pandemic, the following points are worth remarking. plasma should only be collected from selected recovered individuals diagnosed with covid-19 for at least 3 weeks. at least 14 days must have elapsed since complete recovery, 5 biomed research international in order to minimize the possible risk of sars-cov-2 in the blood. the titer of anti-sars-cov-2 igg should be determined, and virus inactivation procedures should be strictly attended before using plasma [5] . actually, the recommended viral neutralization titer cut-off for covid-19 convalescent plasma is at least ≥1 : 160. this corresponds to a receptor binding domain igg titer ≥ 1 : 1350 [35] . a titer of 1 : 80 may be considered acceptable if an alternative matched unit is not available [38] . although largely experimental, the optimal dose of convalescent plasma to be administered to a covid-19 patient ranges between 200 and 500 ml [28, 31-33, 35, 36] . treatment effectiveness is expected to be better when immune plasma is collected from patients of the same city, or surrounding area, since it is assumed that these donors have defeated the same virus (virus genome can mutate); likewise, lifestyle, diet, and environment play an important role in the development of specific antibodies against the virus [39] . as a principle, convalescent plasma should be used as soon as possible in the acute stage of the disease of the recipient [4, 5] , and it is important that the titer of anti-sars-cov-2 antibodies be high. it is essential to ensure abo compatibility between donor and recipient; theoretically, transfusion of plasma from at least two donors may be better to achieve more effective immune protection from delivery of diverse antibodies. standard selection criteria for plasma donation, according to local requirements, must always be followed, as well as standard postdonation treatment of plasma [11] . unfortunately, the brn recommendations have been disregarded. at the beginning of the present pandemic, no healthcare system had already organized programs to collect convalescent plasma or serum from recovered patients to fight a potential new emerging viral pathogen. currently, some clinical studies and programs have started, but unfortunately, the procedures are very slow [7, 40, 41] . the first reported results are very encouraging and confirm the effectiveness of plasma therapy and its safety [42] [43] [44] . the classical process to approve a new drug for clinical use is long, but a terrible pandemic emergency is underway and the time to wrest the fate of many people from death is very short. it is essential that governments follow the strategy recommended by the brn in 2017. recently, the food and drug administration (fda) has also moved on this path, in consideration of the numerous evidences of efficacy and safety of plasma therapy coming from past experiences and the first scientific confirmations in the current pandemic. the administration has remarked the importance to study the safety and efficacy of covid-19 convalescent plasma enrolling patients in clinical trials. in addition, two other strategies have been authorized to allow patients to access treatment. firstly, it provides an expanded access for the use of covid-19 convalescent plasma dedicated to patients with serious or immedi-ately life-threatening covid-19 disease, who are not eligible or unable to participate in randomized clinical trials (21 cfr 312.305) . secondly, given the public health emergency, fda facilitates access to covid-19 convalescent plasma for patients with serious or immediately life-threatening covid-19 infections: the patient's physician can request a single emergency investigational new drug application to obtain expanded access for an individual patient (21 cfr 312.310) [29] . this strategy could allow to save as many lives as possible. in addition, the fda is promoting an awareness campaign to invite patients recovered from covid-19 to donate plasma [45] . expected results from ongoing trials should definitely support the researches in finding the best criteria for including/excluding convalescent plasma in covid-19 patients' treatment. as detailed in the available studies, performed analysis suggests convalescent plasma for the most serious cases and at their early stage. thus, the early recognition of the covid-19 patients who may develop critical illness is the key question for convalescent plasma treatment. they are the patients to be treated with convalescent plasma. it is known that most mild covid-19 patients can be self-recovered, and convalescent plasma may be inappropriate therapy for them. and for end-stage covid-19 patients, the convalescent plasma treatment may not be able to regress the poor outcome as demonstrated by the current studies [46] . the strategy of the fda seems the most correct, since convalescent plasma appears to be a safe and effective therapy, and a vaccine requires a long time to get ready. to date, there are no other authorized therapies against sars-cov-2. furthermore, it should not be forgotten that the other currently applied therapies (e.g., antiviral drugs and hydroxychloroquine) have remarkable side effects and are administered for compassionate use [7, 47] . another key point is that convalescent plasma should be hyperimmune and contain high antibody titers against sars-cov-2. it is still unknown how long patients have good antibody levels in their blood [48] ; therefore, at least hypothetically, the time window for convalescent plasma donation is limited to the first period after a patient's full recovery. fortunately, many patients in the world are recovering from covid-19 infection. this should be the right time to donate plasma to treat seriously ill patients. governments should be aware of this opportunity and start organizing appropriate plasma donation campaigns and adequate plasma collection programs. the author denies any conflict of interest. who pandemia who director-general's opening remarks at the media briefing on covid-19 -11 perspectives on therapeutic neutralizing antibodies against the 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significantly decreased mortality effectiveness of convalescent plasma therapy in severe covid-19 patients a scoping review of registered clinical trials of convalescent plasma for covid-19 and a framework for accelerated synthesis of trial evidence (fast evidence) recommendations for investigational covid-19 convalescent plasma, us fda could intravenous immunoglobulin collected from recovered coronavirus patients protect against covid-19 and strengthen the immune system of new patients? covid-19 convalescent plasma: phase 2 national covid-19 convalescent plasma project using therapeutic plasma exchange as a rescue therapy in covid-19 patients: a case series de donno's plasma therapy seems to work, but nobody cares convalescent plasma transfusion for the treatment of covid-19: systematic review donate covid-19 plasmau.s. food and drug administration site13 maggio effect of convalescent plasma therapy on viral shedding and survival in patients with coronavirus disease 2019 the pharmacological basis of therapeutics. goodman & gilman's, mcgraw-hill education immunity after sars-cov-2 infection key: cord-349031-tbof9yqi authors: chen, shiu-jau; wang, shao-cheng; chen, yuan-chuan title: novel antiviral strategies in the treatment of covid-19: a review date: 2020-08-20 journal: microorganisms doi: 10.3390/microorganisms8091259 sha: doc_id: 349031 cord_uid: tbof9yqi the coronavirus disease 2019 (covid-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (sars–cov-2), is still a global public health problem for humans. it has caused more than 10,000,000 infections and more than 500,000 deaths in the world so far. many scientists have tried their best to discover safe and effective drugs for the treatment of this disease; however, there are still no approved standard therapeutics or effective antiviral drugs on the market. many new drugs are being developed, and several traditional drugs that were originally indicated or proposed for other diseases are likely to be effective in treating covid-19, but their safety and efficacy are controversial, under study, or in clinical trial phases. fortunately, some novel antiviral strategies, such as convalescent plasma, clustered regularly interspaced short palindromic repeats (crispr), and mesenchymal stem cell (msc) therapy, potentially offer an additional or alternative option or compassionate use for the people suffering from covid-19, especially for critically ill patients, although their safety and efficacy are also under study. in this review, we explore the applications, possible mechanisms, and efficacy in successful cases using convalescent plasma, crispr, and msc therapy for covid-19 treatment, respectively. furthermore, the perspectives and limitations of these novel antiviral strategies are evaluated. coronaviruses are enveloped viruses that are spherical in shape and characterized by crown-like spikes on their surface. this type of virus can be further divided into four subgroups: alpha, beta, gamma, and delta. coronaviruses, containing many coronavirus strains, are major pathogens in both humans and other animals. seven strains can cause human diseases, including two alpha coronaviruses (human cov-229e (hcov-229e) and hcov-nl63), five beta coronaviruses (hcov-hku1, hcov-oc43, middle east respiratory syndrome coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sars-cov)), and the newly discovered sars-cov-2, which is responsible for the coronavirus disease 2019 (covid19) pandemic [1, 2] . the genetic element of sars-cov-2 is a positive sense, single-stranded rna (ssrna). the virion has a spirally symmetrical capsid and envelope, and its genome size is about 30 kilobase, which is the largest of all rna viruses. there is a rod-shaped spike glycoprotein on the envelope, which can be connected to the angiotensin-converting enzyme 2 (ace2) receptor on the human cell surface [3] . following the specific binding of the receptor-binding domain of the spike glycoprotein to the ace2 receptor of the host cell, the entry of the viral single-stranded rna genome is facilitated [4] . the host cells begin to synthesize replicases and transcriptases to form a replicase-transcriptase complex, including rna-dependent rna polymerase, rna helicase, rna 5′-triphosphatase, exoribonuclease, n7-methyltransferase, 2′-omethyltransferase, to replicate viral rna and synthesize viral structural proteins. the newly synthesized rna and structural proteins assemble together to form mature virions. finally, the virions are sent to the cell surface in the form of vesicles and then released from the original host cells via exocytosis to infect new host cells ( figure 1 ) [5, 6] . at the beginning of the 21st century, several viral diseases emerged and raised the attention of both medical and public health researchers, including sars from 2002 to 2004, h1n1 influenza in 2009, and mers from 2012 to 2015. recently, covid-19 in wuhan city, in china's hubei province, was first reported as an epidemic to the world health organization (who) on 31 december 2019. who then announced covid-19 as a public health emergency of international concern (pheic) on 12 february 2020. the infection of sars-cov-2 can be brought about by droplet transmission or direct contact with secretions from coughing or sneezing of covid-19 patients and may be followed by clinical symptoms and/or illness. according to the intensity of respiratory dysfunction, covid-19 has a different clinical spectrum, such as uncomplicated (mild) illness, moderate pneumonia, severe who then announced covid-19 as a public health emergency of international concern (pheic) on 12 february 2020. the infection of sars-cov-2 can be brought about by droplet transmission or direct contact with secretions from coughing or sneezing of covid-19 patients and may be followed by clinical symptoms and/or illness. according to the intensity of respiratory dysfunction, covid-19 has a different clinical spectrum, such as uncomplicated (mild) illness, moderate pneumonia, severe pneumonia, acute respiratory distress syndrome (ards), and even sepsis, septic shock, and multiple organ dysfunction syndromes. the main symptoms include fever, dry cough, fatigue, expectoration, shortness of breath, muscle pain or joint pain, sore throat, headache, chills, nausea or vomiting, stuffy nose, diarrhea, coughing up blood, and conjunctival hyperemia. additionally, abnormal smell and/or taste have been reported. most people infected with sars-cov-2 will experience mild to moderate respiratory illness, and some of them will recover without requiring special treatment. however, serious respiratory diseases may follow, including pneumonia in some cases. older people and those with underlying medical problems such as cardiovascular disease, diabetes, chronic respiratory disease, and cancer are a high-risk population and more likely to develop a serious illness, which may even lead to death in some cases [7] . the disease control, diagnosis, treatment, and prevention of covid-19 are very difficult due to the following characteristics of sars-cov-2 [8] . (1) cross-species transmission: it can be transmitted between different species of animals and between people and animals, or among people [8] . (2) diagnostic variability: although there may be a negative diagnosis after repeated tests, it may become positive unexpectedly [8] . (3) multiple transmission routes: it is mainly transmitted through droplets or direct contact, but some studies have even found that it can be transmitted by air [9] [10] [11] [12] , feces [13] [14] [15] , and asymptomatic carriers [16] [17] [18] . moreover, its basic reproductive number (r0) has been determined to range between 2-3 2 , and the spreading region is probably wide enough to trigger a pandemic [19] . (4) genome mutation possibility: the mutation of the viral rna genome is possible [12, 19] ; hence, the research and development of vaccines and drugs are great challenges. even if the discovery is successful and products are approved for marketing, the effective period of the vaccine products and approved drugs may be limited due to mutation and resistance. the high incidence of respiratory failure and high mortality rates result in a heavy burden on medical services, as well as the urgent need of potential medications for covid-19. however, no specific antiviral treatment or vaccine for covid-19 is currently available; that is, there is no evidence from randomized controlled trials to support specific drug treatment for covid-19. at present, the definite therapeutic strategies to deal with the infection are only supportive therapies, which have limited efficacy. for example, symptomatic treatment and oxygen therapy can be used to prevent acute respiratory failure [20, 21] . if oxygen therapy is not helpful, noninvasive and invasive mechanical ventilation (imv), high flow nasal oxygen therapy, and even extracorporeal membrane oxygenation (ecmo) should be considered for the patients with rapidly progressive respiratory failure [22] [23] [24] . regarding traditional ards treatment, gattinoni et al. suggested that covid-19-induced ards is not a "typical" ards [25] , and imv should be postulated earlier to avoid excessive intrathoracic negative pressures and lung injury. the use of systemic corticosteroids for ards or viral pneumonia is not recommended; however, for patients with rapid progression of respiratory failure, methylprednisolone could be considered for use appropriately [26, 27] . if there is any possible accompanying bacterial pneumonia, antibacterial drugs are recommended. despite this, some traditional drugs originally indicated or proposed for other diseases may be effective for the treatment of covid-19. potential therapeutic options of covid-19, such as antimalarials, antivirals against viruses, or cytokines against symptoms, are under study or clinical trials, but their safety and efficacy are still unclear or controversial (table 1 ) [28] . janus kinase 1/2 inhibitor myelofibrosis a number of publications have addressed possible therapeutics of covid-19, but very few reports have confirmed the safety and effectiveness of these therapeutics for the treatment of this disease. no standard treatment for covid-19 has been approved, and most of the traditional drugs for inhibiting sars-cov-2 are still under evaluation. consequently, some novel antiviral strategies can probably be helpful for the treatment of covid-19 and reduction of the death rate of this disease, especially for patients who are at the most dangerous phase of this disease. convalescent plasma is referred to as immune plasma collected from patients who have recovered from infectious diseases. the transfusion of convalescent plasma may contain passive antibodies and provide immediate passive immunity to susceptible individuals for the short-term. meanwhile, the current pandemic of covid-19 and indefinite efficacy of traditional drugs have called attention to convalescent plasma as a novel antiviral strategy to treat this disease. the pathogenesis of covid-19 is probably affected by direct neutralization, control of an overactive immune system (e.g., cytokine storm, th1/th17 ratio, complement activation) by anti-inflammatory and immunoregulatory activity, and immunomodulation of a hypercoagulable state, which are implemented by the components of convalescent plasma [52] . several examples have shown that convalescent plasma has been successfully used as postexposure prophylaxis and/or treatment for covid-19 [53] [54] [55] [56] [57] . although the data are limited, the data suggest clinical benefits in the aspects of radiological resolution, reduction in viral loads, and improved survival in the hope of reducing morbidity and mortality [58, 59] . shen et al. used convalescent plasma transfusion obtained from five patients who had recovered from covid-19 to treat five critically ill patients who had covid-19 and ards [53] . the convalescent plasma contained sars-cov-2-specific antibodies, where the igg-binding titer was greater than 1:1000 and the neutralization titer was greater than 40. although these patients had received antiviral treatment and mechanical ventilation, they still presented severe pneumonia with fast progression and continuously high viral loads [53] . following the receipt of convalescent plasma, three out of five patients were discharged, with the remaining two patients in stable condition at 37 days after transfusion. in this case series study of five critically ill patients with covid-19 and ards, the administration of convalescent plasma containing neutralizing antibodies significantly improved their clinical status [53] . however, the limited sample size and study design preclude a definitive conclusion about the potential efficacy, and these observations are required to be evaluated in clinical trials. duan et al. transfused one dose of 200 ml of convalescent plasma with the neutralizing antibody, derived from recently recovered donors, into 10 patients with covid-19 to explore the possibility of convalescent plasma transfusion to rescue severe patients [54] . after convalescent plasma transfusion, the level of neutralizing antibody remained at a high level (1:640) in nine cases (one case was unavailable). the clinical symptoms and paraclinical criteria were significantly improved within three days. compared with pretransfusion, several clinical data were improved: lymphocyte counts were increased, c-reactive protein was decreased, varying degrees of absorption of lung lesions were shown in radiological examinations, and the viral load was not detected in seven patients who previously had viremia [54] . additionally, there were no serious adverse effects that emerged during this period. these results revealed that convalescent plasma was able to serve as a promising choice for severe covid-19 patients [54] . however, larger, well-controlled, and randomized trials are required for further evaluation of the optimal dose, time point, and clinical benefits. zhang et al. used anti-sars-cov-2 antibodies, including igm and igg measured by enzyme-linked immunosorbent assays (elisas), in convalescent plasma from six donors who had recovered from covid-19 to test if convalescent plasma can be utilized for the treatment of severe covid-19 patients [55] . the recipient was a 64-year-old female. eleven days after convalescent plasma transfusion, they found that she did not require mechanical ventilation and could be transferred to a general ward. the results revealed that the convalescent plasma may facilitate insight into the sars-cov-2 infection and established donor screening protocol for covid-19 [55] . however, the efficacy of convalescent plasma is still indefinite because the favorable outcome was achieved in only one patient (sample size is small), and the clinical outcome may have been confounded by other concomitant treatments. ye et al. did a descriptive study to evaluate the efficacy of convalescent plasma therapy in patients with covid-19 [56] . six patients were enrolled and received convalescent plasma transfusions. the efficacy of convalescent plasma was determined by the alleviation of symptoms, changes in radiologic abnormalities, and laboratory tests. they found that the transfusion of convalescent plasma resulted in a resolution of ground-glass opacities (ggos) and consolidation in five patients, and viruses were removed in two patients. the serologic analysis indicated anti-sars-cov-2 antibody titers immediately increased in two patients. moreover, no severe side effects were observed. the results showed that convalescent plasma was effective, specific, and significant for the removal of sars-cov-2 [56] . however, the study is neither universal nor representative due to the very small sample size. ahn et al., in a case report, used convalescent plasma to treat two patients with covid-19 who presented severe pneumonia with ards [57] . in addition to systemic corticosteroid administration, both patients showed a favorable outcome without any severe adverse effects after convalescent plasma transfusion. the cases suggest that convalescent plasma might be an additional or alternate option to treat patients. moreover, it is possible to simultaneously reduce viral loads by convalescent plasma and an excessive inflammatory response by systemic corticosteroids [57] . however, more scientific evidence and clinical trials are required to prove the efficacy and safety of convalescent plasma. additionally, the given number of antibodies to each patient should be standardized, and other treatments that may influence the correlation between convalescent plasma and antibody should be investigated. the clustered regularly interspaced short palindromic repeats (crispr)/crispr associated protein 9 (cas9) system contains two components: cas9, an endonuclease, and a single-guide rna (sgrna) that guides cas9 to a specific location in the genome. cas9 will unwind the dna duplex and cut both strands as a target sequence is recognized by sgrna. by the cooperation of a specifically designed sgrna and cas9, the genome can be cleaved at most locations with only the availability of a protospacer adjacent motif (pam) sequence (ngg) that is 3 nucleotides upstream from the target site [60] [61] [62] . being a gene-targeting technology, crispr has also been successfully used as an antiviral approach for the elimination of a variety of viruses [60] . therefore, this suggests that the crispr can potentially act as a novel antiviral strategy for the treatment of covid-19. abbott et al. designed and screened crispr-associated rnas (crrnas) to target conserved viral regions and identify functional crrnas targeting sars-cov-2 in cell culture [63] . pac-man (prophylactic antiviral crispr in human cells) is a crispr/cas13-based strategy for rna-guided viral rna inhibition and degradation. in human lung epithelial cells, the rnas from sars-cov-2 sequences and live influenza a virus (iav) can be effectively degraded using pac-man [63] . their bioinformatic analysis revealed that a group of six crrnas could target more than 90% of all coronaviruses. the results demonstrated that pac-man decreased about 90% of viral loads and may be useful for targeting a variety of viruses, and could become a powerful approach in blocking viral replication and gene expression. with the development of a safe and effective delivery system in the respiratory tract, pac-man has the potential to become a crucial strategy for the inhibition of pancoronaviruses [63] . however, this study was only performed in cell culture, and in vivo studies and further clinical trials are needed. cell-based approaches, primarily using mesenchymal stem cells (mscs), have demonstrated to provide a possible safe and effective novel antiviral strategy for patients with ards [64, 65] . immunomodulatory, regenerative, and anti-inflammatory properties of msc treatment have been proposed as a suitable therapeutic approach, and several clinical trials have begun for covid-19 treatment. a set of various cell sources, doses, administration strategies, and target patient populations have been applied in msc therapy [65] . after msc transplantation, the immunomodulatory effect of mscs could protect alveolar epithelial cells, reclaim the pulmonary microenvironment, prevent pulmonary fibrosis, and cure lung dysfunction because of the significant cell accumulation in the lungs [64, 65] . currently, no msc-based therapy has been approved for the prevention and/or treatment of covid-19, but related clinical trials are being continuously conducted now. leng et al. intravenously injected msc into seven enrolled patients to investigate whether msc transplantation is effective in treating covid-19 [66] . they evaluated the patients' clinical outcomes, inflammatory changes, immune function levels, and adverse effects for 14 days. after msc transplantation, all patients' functional outcomes were significantly improved, without any observed adverse effects. the peripheral lymphocytes were increased, and the c-reactive protein was decreased. moreover, the overactivated cytokine-secreting immune cells cxcr3 + cd4 + t-cells, cxcr3 + cd8 + t-cells, and cxcr3 + nk cells disappeared within 3-6 days, and regulatory dendritic cells (dcs) were dramatically increased [66] . compared with the placebo group, the level of tnf-α was decreased and il-10 was increased in the msc treatment group. the clinical outcome of patients was significantly improved, which may have been due to the regulation of inflammatory response and the promotion of tissue repair and regeneration induced by mscs [66] . the results showed that msc transplantation was both safe and effective, especially for critically ill covid-19 patients. atluri et al. described the pathogenesis of coronaviruses, the urgent need for various solutions to covid-19, and the clinical evidence regarding its treatment using stem cells [67] . the role of expanded umbilical cord mesenchymal stem cells (uc-mscs) in treating covid-19 is being studied. the patient's own immune system is critical to curing covid-19. the immune system triggers the production of many inflammatory factors and causes a severe cytokine storm if it is overactivated in the process of destroying viruses. the cytokine storm may impair organs and result in edema, air exchange dysfunction, ards, acute cardiac injury, secondary infection, and may even lead to death. the limited evidence related to uc-mscs in managing covid-19 suggests that compassionate therapy should only be used in critically ill patients to reduce morbidity and mortality [67] . at this stage, the most prospective application of novel antiviral strategies is compassionate use (expanded access) for covid-19, like remdesivir [29] . compassionate use is a possible way for patients with immediate life-threatening conditions or serious diseases to receive investigational medical products, including chemicals, biopharmaceuticals, or medical devices for treatment, free from the limitation of undergoing clinical trials. these investigational medical products have not yet been approved or cleared, and their safety and effectiveness for their specific use have not been confirmed. the investigational medical product may or may not be effective in the treatment of the condition, and their use may cause unexpected and serious side effects. however, under the supervision of an institutional review board (irb) and with the informed consent of patients, medical products being developed can be made available to patients who have no comparable or satisfactory therapy options. compassionate use may be appropriate when the following requirements apply (table 2) [68, 69] . the patient has a serious disease or condition, or whose life is immediately threatened by these disorders. no comparable or satisfactory alternative therapy is available to diagnose, monitor, or treat the disease or condition. it is impossible for patients to enroll in a clinical trial. potential benefit justifies the potential risks of treatment for patients. the investigational medical product will not interfere with clinical trials that could support the development or marketing approval for the indication of this product. patients have serious diseases with no satisfactorily authorized therapies and cannot enter clinical trials. the programs are only put in place if the medicine is expected to help patients with life-threatening, long-lasting, or seriously debilitating illnesses. the medicine must be undergoing clinical trials or have entered the marketing-authorization application process. early studies of the medicine will generally have been completed, though its safety profile and dosage guidelines may not be fully established. convalescent plasma transfusion is considered to be one of the most ancient and well-known therapeutic methods, but it is seldom used for disease treatment due to safety concerns such as rejection, allergy, contamination with microbes, and mixture with nonspecific proteins from donors [70] [71] [72] [73] [74] [75] . moreover, its efficacy in treating diseases is challenging and controversial [70] [71] [72] [73] [74] [75] . recently, with the emergency and severity of the covid-19 pandemic, convalescent plasma has been tried in some critically ill patients to rescue their lives [58, 59, 76, 77] . some successful cases (described in section 3.1) may promote convalescent plasma as a prospective strategy for the treatment of covid-19 [53] [54] [55] [56] [57] . blood centers have been established to be firm infrastructure for collecting and constructing stocks of convalescent plasma to meet the globally growing demand [58] . however, there are still some limitations for the use of convalescent plasma to treat covid-19, as follows [53] [54] [55] [56] [57] [58] [59] [76] [77] [78] : (1) the sample size of the research is too small to be representative; all patients who have successfully used convalescent plasma for therapy are only special and/or specific cases, not universal cases. (2) scientific evidence is not enough because large-scale clinical trials that can represent the target patient populations (e.g., age, race, gender, and comorbid disease) are lacking or still ongoing. (3) the number of antibodies that the patients received was not standardized. (4) the patient usually received convalescent plasma and other treatments (e.g., antiviral agents, steroids) concomitantly. this may have affected the association between convalescent plasma and the antibody, resulting in confusing therapeutic results. (5) the results for patient prognosis and surveillance post-treatment are still unavailable or insufficient; they must be monitored for a long period of time to evaluate the efficacy and side effects carefully. (6) data from rigorously controlled clinical trials are too few, namely, the range of indications (e.g., prevention vs. treatment), as well as an overview of benefits. risk and regulation adaptability need to be considered deliberately. along with the development of a safe and effective gene targeting approach (e.g., crispr) for respiratory tract delivery, pac-man has the potential to become a perspective strategy for pancoronavirus inhibition. the pancoronavirus protection using crispr-cas13d will offer an alternative and complementary strategy over traditional antiviral drugs or vaccines. however, the biggest barrier to clinically utilizing pac-man is the development of in-vivo delivery tools that are safe, efficient, and effective. moreover, there are several technical limitations for using pac-man against sars-cov-2 in clinical applications, as follows [63] : (1) pac-man functions in a cell-autonomous way, suggesting only cells that express cas13d and crrnas are directly protected against the targeted viruses. (2) pac-man needs full expression in a certain percentage of cells, and the range should be determined experimentally prior to use in patients. (3) the competency to adjust cas13d levels and its associated crrnas in cells may be critical to efficiently inhibit viruses, in that viral sequence cleavage efficiency is sensitive to crrna expression. (4) viral genomes may be resistant to inhibition due to the rna secondary structure or protective protein coats; thus, high-throughput screening of crrnas may be needed for the identification of highly effective crrnas targeting live viruses. (5) the risk of mutation or tumorigenesis induced by off-target effects must be avoided and completely removed before undergoing clinical trials [79] [80] [81] . (6) the selection of a suitable delivery tool that is safe, specific, and efficient in delivering crispr to target cells in patients is not easy [82, 83] . the patients would have more unique benefits than traditional antiviral drugs or vaccines when using the pac-man strategy to treat covid-19 if these aforementioned obstacles are overcome. the process of developing new therapeutic strategies and promoting msc-based therapy for clinical application has important and practical implications for the treatment of covid-19. covid-19 is so serious and human lives are valuable; hence, the effectiveness of therapeutic preparation using msc therapy is becoming a hot topic. the msc secretome can provide a novel therapeutic approach for the treatment of covid-19 because of its extensive pharmacological effects, such as anti-inflammation, immunomodulation, regeneration, proangiogenicity, and antifibrosis [84] . based on this evidence, the msc secretome administered by intravenous injection or inhalation is likely to become a promising antiviral strategy for the treatment of covid-19, particularly for patients in critically severe conditions [66, 84] . to maximize potential therapeutic use, it is critical to understand the related preclinical studies and postulated mechanisms of mscs in respiratory lung injuries induced by viruses. though the clinical application of msc therapy to treat covid-19 has not been approved, some promising reports have been recently presented [64, 65] . therefore, stem cell therapy, especially mscs, may be one of the most prospective therapeutics or a combination of treatments for patients with covid-19. however, msc therapy has only been proven to be safe and effective on a specific, special, and limited basis. the case series study, where seven patients were enrolled, has ensured the feasibility and safety of msc therapy, but the clinical efficacy needs to be confirmed in random controlled trials, including more cases, to have significant clinical statistics [66] . moreover, these patients' prognosis should be monitored for the long term to achieve definite evidence of clinical efficacy [66] . the administration and coronavirus task force might hope to use expanded uc-mscs as an evolutionary therapeutic strategy in managing covid-19, with pronged approaches as follows [67] : (1) all agencies should minimize the regulatory burden so that critically ill patients will have access without considering their financial source or support. (2) appropriate safeguards instituted should refrain from negative results from immoral individuals. (3) when patients are in an urgent situation, and with their proper informed consent, the approach can be tried in critically ill patients who have no response to traditional drugs. the covid-19 pandemic has caused serious global economic, political, public health, and even military problems for the past year, and the consequences are currently persisting in many countries. during this period, almost every country has taken measures for disease prevention, such as quarantines, isolation, disinfection, border blockades, traffic cutoffs, social distancing, homestay, and restriction of passenger entry, to prevent a covid-19 epidemic outbreak. there are currently no approved effective drugs or licensed vaccine products for covid-19. under the condition that traditional drugs cannot assure their safety and efficacy for covid-19 treatment, novel antiviral strategies, including convalescent plasma, crispr, and cell therapy, may be able to provide an additional or alternative option or 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bioethical concerns for clinical application gene editing and crispr in the clinic: current and future perspectives delivery approaches for crispr/cas9 therapeutics in vivo: advances and challenges delivering crispr: a review of the challenges and approaches mesenchymal stromal cell secretome for severe covid-19 infections: premises for the therapeutic use we are grateful for the grant support from mackay memorial hospital in publishing this article. the authors declare no conflict of interest. key: cord-284582-xwedgllw authors: korabecna, m.; zinkova, a.; brynychova, i.; chylikova, b.; prikryl, p.; sedova, l.; neuzil, p.; seda, o. title: cell-free dna in plasma as an essential immune system regulator date: 2020-10-15 journal: sci rep doi: 10.1038/s41598-020-74288-2 sha: doc_id: 284582 cord_uid: xwedgllw the cell-free dna (cfdna) is always present in plasma, and it is biomarker of growing interest in prenatal diagnostics as well as in oncology and transplantology for therapy efficiency monitoring. but does this cfdna have a physiological role? here we show that cfdna presence and clearance in plasma of healthy individuals plays an indispensable role in immune system regulation. we exposed thp1 cells to healthy individuals’ plasma with (np) and without (tp) cfdna. in cells treated with np, we found elevated expression of genes whose products maintain immune system homeostasis. exposure of cells to tp triggered an innate immune response (iir), documented particularly by elevated expression of pro-inflammatory interleukin 8. the results of mass spectrometry showed a higher abundance of proteins associated with iir activation due to the regulation of complement cascade in cells cultivated with tp. these expression profiles provide evidence that the presence of cfdna and its clearance in plasma of healthy individuals regulate fundamental mechanisms of the inflammation process and tissue homeostasis. the detailed understanding how neutrophil extracellular traps and their naturally occurring degradation products affect the performance of immune system is of crucial interest for future medical applications. to catch and destroy the infectious microorganisms. these nets are subsequently cleared from the circulation by dnase i and their insufficient clearance can result in occlusion of blood capillaries, leading to impaired microcirculation, enzymatically damaging tissues and further progression of inflammation 15 . the elevated formation of nets was reported in most comorbidities worsening the clinical course of covid-19 16 . the hyperactivated neutrophils and monocytes-macrophages are the usual initiators of the cytokine storm responsible for the most serious consequences of coronavirus sars cov-2 infection 17 . bacteria and protozoa are able to convert the nets using their own enzymes into the dna degradation product such as deoxyadenosine which is toxic for macrophages and causes their apoptosis 18, 19 . despite all these findings and the massive cfdna-based diagnostic technique developments, there are surprisingly few studies focused on the fundamental biological function of cfdna in healthy individuals. one study treated human monocytes either with the plasma of dialyzed patients or healthy individuals, both containing cfdna. only the plasma from dialyzed patients stimulated the production of pro-inflammatory interleukin il 6 in target cells 20 . it was also revealed that the cfdna isolated from individual tumor cell lines or complex tumors injected into animal blood circulation caused tumor transformation, referred to as genometastasis 21 . the biological character and function of unmethylated fetal cfdna was explored and the increased proportion of fetal cfdna in maternal circulation during pregnancy was found 22, 23 .this fetal cfdna stimulated a maternal immune response against the placenta, resulting in the proper timing of labor 22 . the influence of cfdna on human macrophages was tested using isolated cfdna from various blood products with different storage time to emulate the conditions immediately after transfusion. the cells were cultivated in the presence of calf serum bearing its own cfdna. the study found increased expression of genes involved in the innate immune response (iir), including chemokines and their receptors in macrophages, and concluded that cfdna contained in the stored blood products might interfere with the immune system of transfusion recipients 24 . the authors reported the elevated expressions of cxcl8 and ddit3 in experiments, but the results may be affected by the presence of calf serum in all experiments. this raises the following question: what is the fundamental role of cfdna in healthy organisms? we assumed that we could find it by exposing identical cell lines to plasma with and without cfdna and excluding any other factors, such as calf serum or potential damage of cfdna complexes by isolation procedure. we performed all stimulatory experiments using the thp1 cell line as a representative of primary human monocytes 25 to show the fundamental role of cfdna in healthy organisms. the experiments were conducted in duplicates using plasma containing cfdna (np) and the reference one with cfdna removed by dnase (tp) to recognize the effect of plasma cfdna on transcriptome and proteome of monocytes. we used native human plasma samples obtained from healthy volunteers with no animal serum addition to the cultivation medium in order to avoid the presence of uncharacterized animal cfdna and dnases in the experiments. in the discovery phase, we used six plasma samples, searching for differences in transcriptomes related to treatment with np and tp using genechip human gene 2.1 st array strip (thermo fisher scientific). we detected significant differences ( fig. 1a ; supplementary table 1) , which were further validated using single-target quantitative pcr (qpcr) and another ten plasma samples ( fig. 1b-d) . to differentiate between the effects of buffers alone and the effect of dnase turbo treatment, we performed a set of experiments ( supplementary fig. 1 ). the addition of activation buffer containing divalent cations is necessary for the activity of dnase turbo in plasma originally treated with ethylenediaminetetraacetic acid (edta). the addition of this buffer alone led after incubation to the decrease of cfdna in the plasma sample to 57.42% of its original level due to the activation of plasma endogenous dnase i. the incubation of plasma sample with this buffer and dnase turbo resulted in the detection 7.07% of cfdna original amount when measured after its isolation from plasma using qpcr. in this set of stimulation experiments, we tested the effects of the complete procedure allowing the dnase turbo activity in plasma and its subsequent removal but without the addition of dnase turbo itself and compared the results with np and tp samples. all these experiments were performed using a plasma sample obtained from an identical donor. we examined the expression of all validated genes and concluded that the activation of endogenous dnase i was mostly sufficient to produce the basic differences which could be in some cases further strengthened with subsequent dnase turbo treatment ( supplementary fig. 1 ). the procedure serving for the removal of divalent cations was applied during the processing of all treated plasma samples to avoid the elevated concentrations of these ions in samples due to the dnase turbo activation buffer addition. we received the results identical with the results of validation experiments in eight out of eleven examined genes as we explored the stimulatory capacity of only one differentially treated plasma sample. the expressions of didt3 and sesn2 were significantly elevated in cells treated with np samples in validation experiments but decreased in cells treated with np sample of this donor. the ccl24 expression was significantly increased in cells stimulated with tp samples in validation study but elevated when treated with np sample of this individual. the inconsistencies found in the expressions of ddit3, sesn2 and ccl24 may thus reflect the individual variability deserving of further study. we used the validation phase results to perform a direct comparison of signaling pathways activated in cells as a consequence of their treatment with np or tp samples (table 1a , b) using the database reactome. this analysis demonstrated the critical importance of the presence/absence of the intact cfdna for the expression profile of cells and regulatory pathways activation. this fact was also documented by the reactome analysis of results obtained by mass spectrometry used for proteome examination of thp1 cells treated with np or tp (table 1c) . the presented work documented that the cfdna and its clearance in plasma is under physiological conditions indispensable for immune system performance. we demonstrated that monocytes in intact cfdna presence (np) upregulated the central pathways responsible for immune system homeostasis, especially notch signaling 26 | (2020) 10:17478 | https://doi.org/10.1038/s41598-020-74288-2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ and unfolded protein response (table 1a) 27 while the degraded cfdna in plasma (tp) resulted in upregulated cxcl8 expression (table 1b) . this mrna should translate into interleukin 8 (il8), a pivotal protein involved in the direct activation of iir 28 and also regarded as a marker of cellular senescence 29 , but its elevated level was not found in proteomic analysis after our three-hour-long cultivation experiments. nevertheless, we detected the upregulation of proteins contributing to the activation of complement (table 1; supplementary table 2) , confirming the inflammatory state of these cells. the software ingenuity pathways analysis (ipa) was used to explore all obtained data (supplementary table 3 ). the ipa results ( fig. 2a , b) confirmed the findings of reactome analysis (summarized in fig. 2b inset). in thp1 cells cultivated with tp, multiple pathways involved in immune response were detected among significantly changed canonical pathways ( fig. 2a) and overrepresented disease-specific pathways (fig. 2b) . ipa also predicted the accumulation of granulocytes, leucocytes, myeloid cells, phagocytes, and complement activation as the consequence of events signalizing the presence of cells that are confronted mainly with degraded cfdna (fig. 3 ). we developed and tested an experimental workflow which allowed us to compare the effects of cfdna pool in native plasma and cfdna in plasma degraded by endogenous dnase i and additionally with dnase turbo on the thp1 cells. the native plasma samples were fixed by edta as a potent indirect dnase i inhibitor. in the edta treated plasma samples, the activity of the endogenous dnase i is completely stopped 30 . the activation of endogenous dnase i and the subsequent activity of dnase turbo were allowed by addition of an activation buffer containing divalent cations to the edta treated plasma samples. to inactivate these divalent cations, the dnase turbo inactivation procedure was applied according to the protocol provided by the manufacturer for the aqueous solutions of isolated rnas. our results are of course limited by the fact that we are working with such a complex sample as human blood plasma. it is not possible to design the experiments to exclude completely the influence of changing divalent cations concentrations 31,32 during the entire experimental procedure, the presence of cfdna hidden in plasma exosomes 33, 34 or in supramolecular complexes and on cell surfaces 35 . nevertheless, when we quantified the cfdna isolated from np, from the sample exposed to the entire cfdna removal procedure but without addition of dnase turbo and from the tp sample, we found striking gradual decrease in cfdna levels toward the last sample. the changes in expression profiles of selected validated genes were detectable after the decrease of cfdna levels to 69.10% of its original native concentration as the result of endogenous dnase i activity ( supplementary fig. 1 ). therefore we could speculate also about the role of cfdna www.nature.com/scientificreports/ degradation products in the induction of these expression changes. the significance of differences in expression profiles of cells treated with np and tp was statistically proven in validation experiments. we detected namely the differences in immune system regulatory pathways discussed in the next paragraphs. the inflammatory response in mammalian cells is regulated by notch signaling pathways acting through four different notch receptors (notch 1-4) transducing extracellular signals 26 ; the notch 1 is involved in myeloid lineage differentiation leading to monocytes 36 . the constitutive tonic activity of notch signaling pathways was described in non-activated immune cells 26 . we found that the monocytes treated with np expressed hes1 mrna by ≈ 4.63 × more than the ones treated with tp (fig. 1d) . this elevated expression documents the notch pathways' activity in these cells. the pathways leading from toll-like receptors (tlrs) may provide additional signals to the notch ligands 26 . in primary macrophages, it has been shown that the notch target genes, like hes1, can also be induced exclusively by tlr stimulation 37 . in such a model, the presence of cfdna itself could ensure the constitutive tonic notch signaling via tlr9 as a receptor specialized in dna sensing. different types of cellular stress lead to unfolded protein accumulation in the lumen of the endoplasmic reticulum. this accumulation activates signal-transduction cascade known as unfolded protein response (table 1a) . it has been demonstrated that this cascade plays the central role in the modulation of immune system functions regarding both innate and adaptive responses 27 . in our experiments, the expression of sesn2 (sestrin 2) is upregulated in cells treated with np (fig. 1b) . sesn2 is well-known as a stress-inducible protein suppressing inflammasome activation by the induction of mitophagy. sesn2 plays a crucial role in this unique regulatory mechanism of mitophagy activation which is pivotal for the maintenance of immunological homeostasis and protection from sepsis 38 . we found the increased expression of arrdc4 and irf1 genes in cells treated with np (fig. 1b) additionally to the genes involved in the pathways detected by reactome ( table 1 ). the role of arrestin domain-containing 4 (arrdc4) in iir was recognized but only partially understood 39 . this gene is transcribed in monocytes in healthy individuals; after a viral infection, its levels were increased and correlated with concentrations of interleukins in serum. interferon regulatory factor-1 (irf1) is a transcription factor expressed at low levels in immune system cells and induced by different cytokines. it controls the transcription of its target genes in different types of immune cells 40 . cultivation of thp1 cells with tp led to the change of expression pattern. apart from increased cxcl8 expression, we found upregulated expression in mtts1, gpr1, and ccl24 (fig. 1c) . metastasis suppressor-1 (mtss1) was originally identified as a metastasis suppressor in the carcinoma cell line. nowadays, it is known that this multifunctional cytoskeleton scaffold protein regulates the cytoskeleton dynamics and inhibits cell migration 41 . g protein-coupled receptor 183 (gpr183) is a member of the signaling pathway leading to repression of notch signaling 42 . ccl24 codes for eotaxin 2, which is produced by activated monocytes and attracts lymphocytes, basophils, eosinophils, and monocytes to the site of inflammation. it has been reported that eotaxin 2 induced apoptosis of thp1 cells 43 . significantly higher expression of proteins belonging to complement cascade is evident at the proteomic level upon the treatment of cells with tp in comparison with cells treated with np (table 1c ). the soluble complement www.nature.com/scientificreports/ proteins detected in plasma are synthesized mainly in the liver, but their local production by circulating immune cells including monocytes is well described 44 . we documented that the monocytes without contact with physiological cfdna concentrations in plasma activated emergency mechanisms and began to initiate iir. the normal functions of these cells were downregulated (notch signaling), potential migration was inhibited, and the genes for attractants of immune cells (cxcl8 and ccl24) were overexpressed (fig. 2b inset and fig. 3) . previously, we found elevated expression of another key member of the cytokine network, namely tumor necrosis factor-alpha (tnf-α) in tp treated cells 45, 46 . tnf-α functions as a master regulator of inflammation and ensures tissue homeostasis 47 . we reported the statistically significant differences validated in qpcr experiments earlier on two different occasions-in our study in which plasma samples of healthy volunteers were used for stimulation of thp1 cells 45 and in the report studying the stimulatory capacity of plasma samples obtained from patients with celiac disease 46 . under the stringent conditions set by us for the evaluation of genechip experiments, the tnf-α was not reported as differentially expressed, but its expression was higher in cells treated with tp per our previous results. to date, the regulatory mechanisms keeping the cfdna concentrations in plasma at physiological levels are not well understood. natural regulatory mechanisms are balancing the nets production and their clearance. these mechanisms may involve the negative closed feedback loop system, which is widely spread in biology and also used by pharmacologists for drug delivery systems 48 . the failure of the feedback loop mechanism might cause exacerbated dnase i production, resulting in iir. some bacteria are equipped not only with dnases but also with their own 3´-nucleotidases. the activity of these enzymes destructs nets and converts their degradation products into deoxyadenosine which is toxic namely for macrophages and induces their apoptosis 49 . different ectonucleotidases are found on the surfaces of different human cells 50, 51 , their participation in nets degradation and toxic product formation cannot be excluded. our results suggest that the exposition of cells to relatively elevated concentration of cfdna degradation products can evoke and promote the inflammatory state as the consequence of clearance of high cfdna concentrations associated with tissue damage 52 or with the affected clearance of the products of netosis 53 . elevated deregulated netosis is reported in common comorbidities not only in dialyzed patients 54 but it is also typical in most comorbidities characterized as risk factors predisposing to serious complications of sars cov-2 infection 16 . here we provided evidence that the cfdna in human plasma and its clearance represents an essential natural tool for regulation of innate immune response. we used the thp1 cell line as a model of human monocytes to demonstrate that cfdna plays an indispensable role in the immune system homeostasis. the cells treated with the native plasma of healthy volunteers expressed genes whose products maintain immune system homeostasis. however, the cells treated with identical plasma samples with degraded cfdna directly activate iir with elevated production of mrna for interleukin 8 at the transcriptomic level. they also upregulated the complement compounds at the proteomic level. the role of intact and degraded cfdna and their sensing by cells seems to be one of essential aspects of immune system performance; therefore, further studies focusing on this subject are highly topical. it is of utmost importance to understand the mechanisms of cfdna release, the clearance and mechanisms of the homeostasis maintenance, as well as the role of different types of cfdna sequences and their chemical modifications in cfdna mediated regulatory events, in addition to the recognition of all aspects of cfdna presence sensed by the cells. the detailed knowledge of mechanisms involved in immune system regulation by the levels of circulating cfdna may lead to new clinical applications, especially concerning the complete understanding of the pathogenesis of sepsis, covid-19 and therapeutic dnase treatment. monitoring the cfdna level can serve as an actual tool for early well-advanced diagnoses of several diseases such as cancer, sepsis and covid-19, as well as labor timing. this study is the first to show the fundamental role of cfdna and its clearance in plasma of healthy individuals in the regulation of innate immune response, thus warranting further research in this direction. subjects. plasma donors were selected from healthy volunteers who satisfied the following criteria: 1. they were taking no medication 2. they had no chronic illness 3. they had not recovered from an infectious disease in the last two weeks 4. they were in very good physical and psychical status. the subset of samples described in our previous study 45 was utilized for expression arrays experiments. five women and five men aged between 21 and 64 years with age of (35.8 ± 13.8) year (mean ± standard deviation from 10 samples) were selected for validation of quantitative pcr (qpcr) experiments. the ethical committee of the 1st faculty of medicine of charles university and general faculty hospital in prague, czech republic, approved our study. we obtained informed consent from all study participants. all methods were performed per the relevant guidelines and regulations. preparation of plasma samples. whole blood was collected into vacuette tubes containing k3 edta (greiner bio-one). we separated blood plasma using the following steps: centrifugation rate, time and temperature set to 1,800 rpm, 10 min and 4 °c, respectively; then the transfer of plasma into 2 ml low binding collection tubes and centrifugation set to rate of 14,500 rpm for 5 min to remove the residual cells. finally, we transferred the plasma into clean 2 ml dna lobind tubes (eppendorf). the samples were stored at a temperature of − 80 °c. | (2020) 10:17478 | https://doi.org/10.1038/s41598-020-74288-2 www.nature.com/scientificreports/ before cell experiments, the plasma sample thawed and split into two identical aliquots. the first one was used at its native state (np), and the second one was treated with turbo dna-free dnase (thermo fisher scientific) according to the manufacturer's recommendation (tp). each 100 μl of the plasma sample was treated with 1 μl turbo dnase, 7 μl water and 12 μl 10 × turbo dnase buffer. this mix was incubated at 37 °c for 20 min and then turbo dnase was inactivated by adding 12 μl dnase inactivation reagent, mixed and incubated at room temperature for 5 min. dnase inactivation reagent was removed by centrifugation at an acceleration force of 10,000 g for 1.5 min and the supernatant was transferred into the new tube. in order to evaluate the influence of individual protocol steps on the alteration of expression profiles, the following experiments were performed: the plasma sample was handled according to the protocol for dnase turbo treatment but dnase turbo was supplemented by water to recognize the effect achieved by this enzyme. inactivation buffer was added either immediately or after incubation step to recognize the influence of sample incubation at 37 °c with activation buffer. identical experiments were performed with dnase turbo addition. all these plasma samples were used for stimulation experiments with thp1 cells and expressions of all validated genes was determined as described below. for determination of cfdna levels in plasma before and after treatment with turbo dna-free kit components, the qpcr at quantstudio 12 k flex real-time pcr system (applied biosystems, usa) was performed using the cfdna samples isolated by qiaamp circulating nucleic acid kit. powerup sybr green pcr master mix (life technologies, usa) constituted one half of 20 μl reaction, the total cfdna amount was measured using primers for the gene 36b4 45 and the standard curve dilution had range from 5 ng to 0.312 ng per reaction. cell cultivation and stimulation. we used the thp1 cell line for stimulation experiments. after the recovery of cells from a deep-frozen state, we used the protocol published earlier 45, 46 . the cells were stimulated for 3 h in the rpmi 1640 medium (sigma aldrich) containing 10% of a plasma sample. the cells were collected after 3 h of cultivation; after centrifugation, the supernatant was removed, and the cells were preserved in lysis solution (sigma-aldrich) and stored at − 80 °c. the results were multiplied by a factor of 10 3 to increase resolution and presented in arbitrary units (au). statistical analysis was done in graphpad prism 5.00.288 software (graph-pad software). first, we performed the d´agostino-pearson normality test. in the case of parametric data distribution, the t-test was used; the wilcoxon matched-pairs signed-rank test was applied for non-parametric data distribution. the statistical significance was set to level ≤ 0.05 for all comparisons. sample preparation for mass spectrometry. frozen thp-1 cell pellets containing ≈ 500,000 cells were resuspended in 100 μl p phosphate-buffered saline and lysed using buffer composed of 1% sodium dodecyl sulfate (sigma-aldrich) in 50 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer with ph 8.5 (carl-roth) supplemented with 1× complete ultra protease inhibitor cocktail-ethylenediaminetetraacetic acid (roche) and shortly sonicated. mixtures were heated for 5 min at 95 °c and subsequently cooled by placing samples on ice. twenty-five units of benzonase nuclease (sigma-aldrich) was added into each tube, and the tubes were incubated at 37 °c for 30 min to degrade chromatin. the obtained protein mixtures were centrifuged to remove cell debris, and the supernatant was determined by the bca method using the nanodrop uv-vis spectrophotometer (thermo fisher scientific). | (2020) 10:17478 | https://doi.org/10.1038/s41598-020-74288-2 www.nature.com/scientificreports/ proteins in the lysate (25 μg) were reduced in the protein lobind tubes (eppendorf) by the addition of dithiothreitol solution (sigma-aldrich) to a final concentration of 10 mm and incubated for 30 min at 45 °c. alkylation was performed by the addition of iodoacetamide to a final concentration of 40 mm and incubation for 30 min at 25 °c in the absence of ambient light. reactions were quenched by the addition of 1 μl of 1 m dithiothreitol per tube. then the protein solution was acidified by 1% formic acid to reach ph value between 2 and 3. in the same tube, 50 μg of suspension of paramagnetic carboxylate-modified microparticles sera-mag speedbeads 1 μm (ge healthcare) was resuspended in the protein sample solution. acetonitrile was immediately added to obtain fifty percent solution, and sample suspension was mixed and incubated for 20 min at 25 °c. next, the tube was placed into a magnetic stand, and paramagnetic microparticles with captured proteins were washed twice using 1 ml of 70% ethanol for 30 s. finally, microparticles were dried by 200 μl of acetonitrile for 15 s and resuspended in ≈ 50 μl of ≈ 50 mm triethylammonium bicarbonate buffer ph 8.0. proteins were eluted by on-bead digestion after the addition of trypsin/lys-c protease mixture (promega) in enzyme to substrate ratio of 1:35 and overnight incubation at 37 °c 55 . the next day, the obtained peptide eluate was discharged from microparticles, which were again washed twice using 25 μl of 25 mm triethylammonium bicarbonate ph 8.0. the pooled peptide mixture was acidified by 4 μl of 10% trifluoroacetic acid and then desalted using omix c18 pipette tips (agilent) according to the user manual. the clean peptide sample was evaporated on the vacuum concentrator (eppendorf) and stored at − 80 °c in protein lobind tubes. nanolc-ms analysis. nano reversed-phase columns (easy-spray column, 50 cm × 75 µm id, pepmap c18, 2 µm particles, 10 nm pore size) were used for liquid chromatography/mass spectrometry analysis. mobile phase buffer a and b was 0.1% formic acid in water and acetonitrile, respectively. samples were loaded onto the trap column model c18 pepmap100 with 5 μm particle size and dimension of 300 μm × 5 mm (thermo scientific) for 4 min at 17.5 μl min −1 . the loading buffer was composed of water, 2% acetonitrile and 0.1% trifluoroacetic acid. peptides were eluted with the mobile phase b gradient from 4 to 35% b in 120 min. eluting peptide cations were converted to gas-phase ions by electrospray ionization and analyzed on a thermo orbitrap fusion modelq-ot-qit (thermo fisher scientific). survey scans of peptide precursors from 350 to 1400 m z −1 were performed at 120 k resolution at 200 m/z with a 5 × 10 5 ion count target. tandem ms (ms 2 ) was performed by isolation at 1.5 th with the quadrupole, hcd fragmentation with a normalized collision energy of 30, and rapid scan ms 2 analysis in the ion trap. the ms 2 ion count target was set to 10 4 , and the maximum injection time was set to 35 ms. only those precursors with charge states from 2 to 6 were sampled for ms 2 . the dynamic exclusion duration was set to 45 s with a 10 ppm tolerance around the selected precursor and its isotopes. monoisotopic precursor selection was turned on. the instrument was run in top speed mode with 2 s cycles 56 . ms 2 data analysis. all data were analyzed and quantified with the maxquant software (version 1.6.1.0) 57 . the false discovery rate (fdr) was set to 1% for both proteins and peptides, and we specified a minimum peptide length of seven amino acids. the andromeda search engine was used for the ms 2 spectra search against the human as downloaded from current uniprot human database. enzyme specificity was set as c-terminal to arg and lys, also allowing cleavage at proline bonds and a maximum of two missed cleavages. dithiomethylation of cysteine was selected as fixed modification and n-terminal protein acetylation and methionine oxidation as variable modifications. the match between runs feature of maxquant was used to transfer identifications to other lc-ms 2 runs based on their masses and retention time with a maximum deviation of 0.7 min; this was also used in quantification experiments. quantifications were performed with a label-free algorithm in maxquant41 58 . data analysis was performed using perseus 1.6.2.2. software 59 . bioinformatic analysis. the sets of differentially transcribed genes and differentially expressed proteins were analyzed using reactome dababase 60 . the sets were also subjected to ingenuity pathway analysis (qiagen) 61 . studies on the chemical nature of the substance inducing transformation of pneumococcal types: induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type iii comptes rendus des seances de la societe de biologie et de ses filiales modifications transmitted to the offspring, provoked by heterograft in'solanum melongena' a historical and evolutionary perspective on the biological significance of circulating dna and extracellular vesicles characteristics of a soluble nuclear antigen precipitating with sera of patients with systemic lupus erythematosus presence of fetal dna in maternal plasma and serum liquid biopsies: genotyping circulating tumor dna genomics-based non-invasive prenatal testing for detection of fetal chromosomal aneuploidy in pregnant women cell-free dna (cfdna): clinical significance and utility in cancer shaped by emerging technologies validation of a clinical-grade assay to measure donor-derived cell-free dna in solid organ transplant recipients cell-free dna comprises an in vivo nucleosome footprint that informs its tissues-of-origin the virtosome-a novel cytosolic informative entity and intercellular messenger high-resolution profiling of fetal dna clearance from maternal plasma by massively parallel sequencing increase in and clearance of cell-free plasma dna in hemodialysis quantified by real-time pcr deoxyribonuclease is a potential counter regulator of aberrant neutrophil extracellular traps formation after major trauma sars-cov2 may evade innate immune response, causing uncontrolled neutrophil extracellular traps formation and multi-organ failure clinical, molecular and epidemiological characterization of the sars-cov2 virus and the coronavirus disease 2019 (covid-19), a comprehensive literature review staphylococcus aureus degrades neutrophil extracellular traps to promote immune cell death cloning, expression and purification of 3'-nucleotidase/nuclease, an enzyme responsible for the leishmania escape from neutrophil extracellular traps apoptotic cell-free dna promotes inflammation in haemodialysis patients origins, structures, and functions of circulating dna in oncology cell-free fetal dna-a trigger for parturition cell-free fetal dna in maternal plasma during physiological single male pregnancies: methodology issues and kinetics cell-free nucleic acids are present in blood products and regulate genes of innate immune response establishment and characterization of a human acute monocytic leukemia cell line (thp-1) role of notch signaling in regulating innate immunity and inflammation in health and disease the unfolded protein response in homeostasis and modulation of mammalian immune cells role of the cxcl8-cxcr1/2 axis in cancer and inflammatory diseases oncogene-induced senescence relayed by an interleukin-dependent inflammatory network edta-mediated inhibition of dnases protects circulating cell-free dna from ex vivo degradation in blood samples modulating macrophage polarization with divalent cations in nanostructured titanium implant surfaces divalent cation signaling in immune cells new evidence that a large proportion of human blood plasma cellfree dna is localized in exosomes mechanisms of nuclear content loading to exosomes generation of blood circulating dnas: the sources, peculiarities of circulation and structure distinct and regulated expression of notch receptors in hematopoietic lineages and during myeloid differentiation integrated regulation of toll-like receptor responses by notch and interferon-gamma pathways sesn2/sestrin2 suppresses sepsis by inducing mitophagy and inhibiting nlrp3 activation in macrophages arrdc4 regulates enterovirus 71-induced innate immune response by promoting k63 polyubiquitination of mda5 through trim65 irf family of transcription factors as regulators of host defense mtss1: a multifunctional protein and its role in cancer invasion and metastasis g protein-coupled receptor 183 facilitates endothelial-to-hematopoietic transition via notch1 inhibition eotaxin-2 induces monocytic apoptosis in patients who have undergone coronary artery bypass surgery and in thp-1 cells in vitro regulated by thrombomodulin production of complement components by cells of the immune system cell-free dna from human plasma and serum differs in content of telomeric sequences and its ability to promote immune response immunoregulatory properties of cell-free dna in plasma of celiac disease patients-a pilot study checkpoints in tnf-induced cell death: implications in inflammation and cancer advances in bioresponsive closed-loop drug delivery systems infection: microbial nucleases turn immune cells against each other nucleotidase activities in soluble and membrane fractions of three different mammalian cell lines the ectonucleotidases cd 39 and cd 73: novel checkpoint inhibitor targets neutrophil dysfunction, immature granulocytes, and cell-free dna are early biomarkers of sepsis in burn-injured patients: a prospective observational cohort study molecular mechanisms regulating netosis in infection and disease netosis provides the link between activation of neutrophils on hemodialysis membrane and comorbidities in dialyzed patients ultrasensitive proteome analysis using paramagnetic bead technology the one hour yeast proteome maxquant enables high peptide identification rates, individualized ppb-range mass accuracies and proteomewide protein quantification accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed maxlfq the perseus computational platform for comprehensive analysis of (prote) omics data the reactome pathway knowledgebase causal analysis approaches in ingenuity pathway analysis m.k. designed the experiments, interpreted the results and wrote the manuscript; a.z. and i.b. performed preparation of plasma samples, cell cultivation and qpcr experiments; b.c. performed microarray experiments; p.p. conducted proteomic analysis and data interpretation; l.s. and o.s. conducted ipa analysis; p.n. was involved in results interpretation and manuscript writing. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-74288 -2.correspondence and requests for materials should be addressed to m.k.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-294684-wfsdjs1f authors: vesnaver, elisabeth; goldman, mindy; o’brien, sheila; macpherson, paul; butler-foster, terrie; lapierre, don; otis, joanne; devine, dana v.; germain, marc; rosser, andrew; macdonagh, richard; randall, taylor; osbourne-sorrell, william; clement-thorne, broderic; al-bakri, taim bilal; rubini, kyle a.; hill, nolan e.; presseau, justin title: barriers and enablers to source plasma donation by gay, bisexual and other men who have sex with men under revised eligibility criteria: protocol for a multiple stakeholder feasibility study date: 2020-11-02 journal: health res policy syst doi: 10.1186/s12961-020-00643-4 sha: doc_id: 294684 cord_uid: wfsdjs1f background: blood donation policy in canada for gay, bisexual and other men who have had sex with men (gbmsm) has changed progressively in the last decade from indefinite deferral to 3-month deferral from last male-to-male sex. driven by safety data and overseen by the national regulator, more inclusive policies continue to redress the disparity in donation for gbmsm. at the same time, the need for source plasma to prepare fractionated blood products is growing worldwide. the collection and processing of source plasma ensures greater safety compared to whole blood donation with respect to transfusion-transmitted infection. this greater safety offers an opportunity to evolve policies for gbmsm from time-based to behaviour-based deferral using revised eligibility criteria. however, changing policies does not in itself necessarily guarantee that gbmsm will donate or that staff in donor clinics are ready to support them to do so. in anticipation of a move to behaviour-based donation screening for gbmsm in canada, we aim to assess the acceptability of and perceived barriers and enablers to source plasma donation using revised screening criteria for gbmsm among key stakeholders to inform policy implementation strategies. methods: this mixed-methods feasibility study will involve gbmsm and donor centre staff to understand modifiable barriers to implementing more inclusive eligibility criteria. key informant interviews and surveys will be rooted in the theoretical domains framework to identify modifiable factors associated with source plasma donation motives in gbmsm and training needs in donation centre staff. we will use an integrated knowledge translation approach involving a partnership between researchers, the national blood operator and gbmsm, situating knowledge users as key research team members to ensure their perspectives inform all aspects of the research. discussion: our integrated knowledge translation approach will provide a more comprehensive and collaborative understanding of blood operator and gbmsm needs while accelerating the implementation of study findings. given the historical backdrop of the decades of exclusion of sexually active gbmsm from blood donation, this study has the potential not only to inform a process and policy for gbmsm to donate source plasma, a blood product, but also offers opportunities for new relationships between these knowledge users. the demand for plasma proteins (e.g. immunoglobulins) continues to rise globally, outstripping the supply. canada does not collect enough source plasma to meet the needs of its citizens and would benefit from widening the range of possible donors, especially to those who may be interested but limited from doing so, such as gay, bisexual and other men who have sex with men (gbmsm). source plasma is a type of plasma donation that is frozen and then sent to a manufacturer for the production of specific plasma protein products such as intravenous immunoglobulins. the additional processing involves pathogen reduction and additional assurance of supply safety compared to whole blood donation with respect to transfusion-transmitted infection. this greater safety offers an opportunity to widen the range of possible source plasma donors with more inclusive eligibility screening. in the early 1980s, in canada and many other countries, blood donation by gbmsm was banned based on the higher seroprevalence of hiv in this group and because there was no test available to detect hiv infection at the time [1] . donor criteria have moved progressively from a permanent deferral (cannot donate) to a 3-month deferral since the last time of sexual encounter with a goal of moving to behavioural risk screening for gbmsm donors. the risk of hiv infection is not the same for all gbmsm; behavioural risk screening would enable identification based on sexual behaviour rather than time since sexual encounter [2, 3] . currently, canadian blood donor criteria do not permit any sexually active gbmsm to donate whole blood or source plasma-men who have had sex with a man in the past 3 months are deferred. however, canadian blood services, one of canada's two national blood operators, is reviewing its policies regarding source plasma donation in an effort to be more inclusive while maintaining the safety of the blood supply, including some gbmsm that are at low to no risk of hiv infection such as those in monogamous relationships. it is likely that many in the general population, including gbmsm, are not yet as aware of what source plasma donation entails and how it differs from whole blood donation. source plasma donation involves drawing whole blood, separating out the plasma, and then returning red and white blood cells and platelets back to the donor. the source plasma donation process takes approximately 45 min and can be donated as frequently as every week. once collected, source plasma can be frozen and stored for months and the manufacture of plasma protein products includes several pathogen reduction steps to kill any residual infectious agents. conversely, fresh components collected during whole blood donation, such as platelets and red blood cells, have a short storage period of up to 7 or 42 days, respectively, and are currently not subjected to the same pathogen reduction processes as source plasma in canada. both the longer storage time of source plasma before pooling and the pathogen reduction manufacturing processes involved in the production of plasma protein products make it feasible to apply additional safety steps that may allow some sexually active gbmsm to donate source plasma. expanding the eligibility to gbmsm for source plasma donation first provides an opportunity to collect data on gbmsm donors whilst maximising the continued safety of the supply. data on gbmsm donors is critical to support changes to whole blood screening policies; with current deferrals in place and gbmsm unable to donate, no such data can be collected. as of 2019, all male donors are asked 'in the last 3 months, have you had sex with another man?' responding yes to this question results in a 3-month deferral from last sexual contact, which amounts to an indefinite deferral for a donor in an ongoing relationship. revised eligibility criteria could include adding additional behavioural screening questions to those who answer 'yes' to identify gbmsm that are at low to no risk of hiv infection. the additional behavioural questions under consideration include: 1 have you had sex with a new partner in the last 3 months? 2 have you had sex with more than one partner in the last 3 months? 3 are you in a mutually exclusive (monogamous) relationship? these questions are based on those in use in jurisdictions such as france (quarantined plasma programme), italy and spain [4] [5] [6] as well as data generated in the ongoing canadian research studies on gbmsm and blood donation. the use of the draft questions in this study will permit further validation of their clarity and acceptability, which may lead to modifications in the actual questions submitted to health canada for implementation in a source plasma collection site. making donation possible is necessary but likely insufficient for a successful gbmsm plasma donation programme. given the historical exclusion of gbmsm from any blood or blood product donation, any gbmsm donation programme would require general acceptability among gbmsm. in canada, caruso et al. [7] explored the acceptability among gbmsm of a plasma donation programme involving quarantining plasma until donors return 2 months or more after initial plasma donation and are retested for transmissible diseases, thus ensuring the safety of the supply prior to release. the programme was considered by some participants to reinforce the exclusion and discrimination experienced with whole blood deferral policies because gbmsm donors and their plasma are treated differently. while behavioural risk screening is an approach that has previously been supported by gbmsm with respect to whole blood donation [8, 9] , there is also a need to explore how gbmsm understand and would respond to the screening questions. studies of gbmsm that have donated blood despite not being eligible reported that participants donated because they were uninformed about the policies or they assessed their own hiv risk to be low with a mixed understanding of the 'window period' , the period in which new hiv infection is not detectable but potentially infectious [8] [9] [10] . exploring how gbmsm understand the behavioural screening questions prior to implementation can help uncover and address the challenges of adherence to the policies. the ineligibility of sexually active gbmsm from blood and plasma donation has attracted debate worldwide with regards to justice, exclusion, safety, risks and community [11] [12] [13] [14] . time-based deferrals that involve policies specific to gbmsm have been described by some as discriminatory, disproportionate to risk and unnecessarily strain the supply of available blood (and plasma) products [15, 16] . the gbmsm blood donor debate has ignited many gbmsm and allies to equate blood donation with equality and full citizenship and has resulted in lawsuits, boycotts of blood drives and petitions [16, 17] . the effects of group exclusion from blood donation can be long lasting as has been observed in the haitian-canadian community [18] . an extension of existing donation promotion strategies without such considerations is likely to miss the mark. understanding how gbmsm view the historical context and changes of the policies in relation to how they view the proposed behavioural risk screening approach will generate insights for the development of appropriate strategies for communication of the policy and promotion of source plasma by gbmsm. the staff in donation clinics who would be tasked with implementing the revised eligibility criteria for gbmsm play a central role in the safety and success of such a programme. alteration of the eligibility criteria may require additional training and staff may have views and perspectives that, if surfaced early, would help to ensure a feasible and acceptable roll-out of a gbmsm source plasma donation programme. indeed, hughes et al. explored the views of blood collection organisation staff on the transition from indefinite deferral to 12-month deferral in gbmsm in california [19] . they showed that, while some staff voiced reservations, most staff supported this transition. they also showed that staff valued their professionalism and would follow the regulations imposed by the food and drug administration in the united states regardless of their own personal views. furthermore, most staff expressed a desire for further training and materials. findings such as these highlight the importance of understanding the potential barriers and enablers to the implementation of revised eligibility criteria to ensure that such a change could be feasibly implemented. however, the hughes et al. study focused on whole blood donation and the move from indefinite to 12-month donation deferral in gbmsm. there is a need to explore such views in more detail in a canadian setting for source plasma donation and for revised eligibility criteria that moves away from a time-since-sexual-encounter criterion. while this is a promising opportunity for more inclusive donation to enhance the donor pool with a previously excluded segment of the population, it is important to assess the feasibility with key knowledge users prior to implementation. the proposed study is designed to fill this gap and provide information from gbmsm and donor centre staff on whether revised eligibility criteria for gbmsm is viewed to be feasible and acceptable and what factors could be adapted to enhance these features [20] . the implementation of revised eligibility criteria and gbmsm source plasma donation can be described as an intervention designed to support behaviour change in gbmsm. due to the number of different knowledge users that may each require different targeted strategies, the intervention can be described as a 'complex' intervention. the united kingdom medical research council guidance on the development and evaluation of complex interventions highlights the importance of careful intervention development using appropriate theory and feasibility assessment prior to evaluation and implementation [21] . although there are extant theories to draw from to understand first time blood donation (as it would be for most gbmsm), the application of behavioural science to source plasma donation is scant and, in canada, source plasma donors are typically recruited from among whole blood donors [22] . furthermore, the historical backdrop of exclusion of gbmsm from donation may have resulted in drivers that are unique to this population. a systematic approach to intervention development can provide transparency and foster cumulative evidence to ensure that the strategies devised to support gbmsm in donating and staff in screening are fit for purpose. french et al. [23] proposed a four-step approach for designing theory-based implementation interventions that suggests key steps in the systematic development of an intervention to change behaviour as follows: first, identify who needs to do what differently; second, identify what barriers and enablers might be relevant; third, select change strategies and techniques that are fit for purpose to address identified barriers and enablers; and fourth, identify how change can best be measured. this study will focus on completing step two using a comprehensive theoretical framework. to ensure the findings are useful to all knowledge users, this study will be rooted in an integrated knowledge translation (ikt) approach, a form of research coproduction [24] . research co-production is an equitable collaborative approach to research that meaningfully engages knowledge users who are directly impacted by the results of the research [25] . ikt is distinguished from other co-production approaches with its emphasis on collaboration with knowledge users that are in positions of power to create change [26] , the focus of study is identified by knowledge users, and research focuses on "generating real-life solutions to complex problems" [27] . the purpose of this study is to assess the feasibility and acceptability of source plasma donation with revised eligibility criteria for gbmsm in two canadian cities. our specific objectives are to identify the following: 1 views and experiences of gbmsm regarding source plasma donation, current eligibility criteria and revised eligibility criteria. 2 the acceptability of additional behavioural questions during the screening process from the perspective of gbmsm. 3 potential barriers and enablers to source plasma donation from the perspective of gbmsm. 4 the acceptability of additional behavioural questions during the screening process from the perspective of donor centre staff. 5 potential barriers and enablers from the perspective of donor centre staff in the donation clinic to implementing new eligibility criteria for gbmsm to donate and to inform the adaptations needed to centre flow and processes prior to piloting. 6 consistencies and discrepancies between the two canadian cities and implications for tailoring strategies to support gbmsm source plasma donation in each context. consistent with an ikt approach, the initial topic was born out of canadian blood services seeking to better understand the feasibly of a source plasma programme for gbmsm with more inclusive donation eligibility criteria and the broader voice of gbmsm demanding greater equality as it relates to donation. the research questions were collaboratively developed by the research team consisting of scientists and collaborators from research institutions, canada's two national blood operators, and a local lgbt2q+ community organisation to ensure diverse perspectives and expertise. while our team offers both insider and outsider perspectives with respect to the donor centre working environment and identifying as gbmsm, we sought out greater inclusion of gbmsm voices in the design and conduct of the study design. we first consulted with community members through outreach and engagement activities and these activities resulted in the formation of a local advisory group of gbmsm. local advisors are key members of the research team (rather than participants) who provide ongoing input in monthly group meetings and in individual exchanges by email or phone as needed. they provide feedback on study design, contribute to survey and interview guide development, and help to develop and facilitate the recruitment strategies. they will review and provide feedback of summaries of analysis and results, advise on next steps, and help disseminate findings. this mixed-methods feasibility study will explore the views of gbmsm and donor centre staff regarding source plasma donation and eligibility criteria to better understand the modifiable barriers and enablers to implementing revised eligibility criteria. we will use qualitative interviews and an online anonymous survey to identify the barriers and enablers to source plasma donation that may emerge with the implementation of revised screening criteria for source plasma donation by gbmsm. qualitative interviews will be used to elicit potential barriers and enablers to implementing revised eligibility from the perspectives of donor centre staff. this study will be conducted in london (ontario) and calgary (alberta). canadian blood services operate two dedicated source plasma donation centres that are located in london (ontario) and calgary (alberta). these centres are potential locations for a first implementation of a gbmsm source plasma donation programme if approved by health canada. the study was first developed in london (ontario) due to operational feasibility and strong relationships with the local gbmsm community. the research team sought additional funding to expand the project to calgary (alberta). many factors may emerge as barriers and enablers to source plasma donation by gbmsm or to donor centre staff 's implementation of revised eligibility criteria. solutions and strategies for encouraging and supporting donation should be tailored to address these barriers to ensure that the supports developed are fit for purpose [28] . theories of behaviour provide a useful set of factors to consider when investigating barriers and enablers. by providing an understanding of which modifiable factors may be associated to implementing revised criteria, such theories provide a source of factors that could then be directly targeted to develop strategies and materials to support donation. there are many different theories that could be used as a basis for identifying such factors in a systematic way. a group of researchers sought to synthesise key content across 33 predominant theories and the 128 constructs within them. they developed the theoretical domains framework (tdf) [29, 30] , which summarises key factors from theory that are known to be associated with behaviour and behaviour change and is well suited to explore the full breadth of factors that are relevant in this behaviour and population. the tdf identifies 14 different modifiable factors, as follows: knowledge, skills, beliefs about capabilities, optimism, beliefs about consequences, intention, goals, professional/social role and identity, social influences, reinforcement, behavioural regulation, emotion, memory/attention/decision processes, and environmental context and resources. clear guidance has been developed to use the tdf for developing qualitative interview guides [31] and quantitative surveys [32] . the tdf has been used broadly as a basis for understanding barriers and enablers in the healthcare setting and with the public [32] [33] [34] [35] . once identified, this approach specifically suggests particular strategies and behaviour change techniques that best suit addressing the barriers and enablers identified based on expert review and the evidence base [36] . participants and recruitment we will use a combination of purposive and snowball sampling to recruit adult (18+) gbmsm in london (ontario) and calgary (alberta) as well as in surrounding communities for interviews. purposive sampling will be used to recruit gbmsm who represent a breadth of age, cultural backgrounds and geographic locations (rural/urban). we will work with our local advisory groups, local organisations and social groups that provide services to gbmsm to help identify potential participants. in 2020, many countries, including canada, were practicing social distancing to reduce the spread of covid-19. in light of this, we will advertise on social media platforms with assistance from organisations that provide services to gbmsm. we recognise that this method of sampling may not reach those who are not active on social media and, as such, we will supplement this strategy with snowball sampling methods by inviting participants to recommend others for participation. snowball sampling is well suited for the recruitment of traditionally underserved groups and those who experience stigmatisation, including gbmsm. procedure we will conduct up to two semi-structured interviews per participant, by phone, scheduled over a period of 2-6 weeks. informed consent will be obtained prior to the interview. interviews will be approximately 60 min in length and audio-recorded for verbatim transcription. the use of a multiple interview format will promote the development of rapport over time, facilitating the discussion of sensitive and personal topics while allowing time for reflection and elaboration [37] . we will offer the option of one longer interview if participants prefer, to take a participant-centred approach to data collection. field notes will be captured after each interview to assist with analysis and reflection on the impact of the interviewer's positionality on the data generated. interview participants will receive a cad$ 20 gift card at each interview session to thank them for their time, to a maximum of cad$ 40 per person. participants that opt for one longer interview will receive a cad$ 40 gift card. interview guide development the first interview will explore the context of how source plasma donation is perceived by gbmsm using a semi-structured approach to interviewing. key interview questions will be used to help define the areas to be explored but the interview style will remain flexible to allow for the discovery and discussion of topics of importance to the participant [38, 39] that may not have otherwise been thought of as pertinent by the research team [40] . the topic guide includes questions regarding experiences of donation, deferral or exclusion, views on current gbmsm donor deferral criteria, and the acceptability of the three behavioural screening questions suggested for inclusion in revised eligibility criteria. the second interview will build on the first to elaborate on emerging themes and explore participants' views regarding the implementation of revised eligibility criteria, potential impacts of revised eligibility criteria on donation practices, and possible barriers and enablers to source plasma donation. the topic guide for the second interview draws on existing literature [31] and previously developed guides [33] to assess if and how the identified barriers align with tdf domains. interview guides will be reviewed by the study's local advisory groups in each city and piloted prior to broad enrolment. at the end of the interviews, participants will be asked about the interview experience and this feedback will be considered and incorporated as appropriate. sample size our sample size will be determined by the available number of interviewees. we aim to recruit 15-20 men from each region to complete a series of two interviews. this sample size estimate is informed by the scope of the study, the nature of the topic and the use of a multiple interview format [41] . given that data collection and analysis are concurrent, informational and thematic redundancy [42, 43] will be assessed on an ongoing basis and the number of interviews will largely be driven by the quality and richness of data [44] . drawing on the literature, saturation is often reached within 15-20 tdf interviews [33, 34] . data analysis interviews will be audio recorded, transcribed verbatim and de-identified prior to analysis. as an additional step, we will send each participant their written transcript to review for completeness and resonance. we will use nvivo12 to facilitate analysis. data collection and analysis will be concurrent to generate emerging understanding of the research questions, which will inform both the sampling and the questions being asked [39] . we will conduct two phases of qualitative descrip-tive analysis-inductive thematic analysis [45] to identify, analyse and report patterns within the data, and theorydriven, directed content analysis to code barriers and enablers expressed by gbmsm to specific tdf domains [46] . throughout, reflexive journaling will be used to capture the analytic process and any developing insights about the patterns in the data. data from each region will be analysed separately as each context may have unique cultural, societal and political forces that shape the views of gbmsm regarding source plasma donation. we will contrast findings between cities to inform city-specific modifications for implementation. during thematic analysis, we will follow the six analytic steps proposed by braun and clark [45] : becoming familiar with the data, generating initial codes, searching for themes, reviewing themes, defining and naming themes, and producing the report. we will then re-visit the data during directed content analysis and use guidance from the literature [31] to code barriers and enablers to source plasma donation expressed by gbmsm to specific tdf domains [46] . we will develop a code book to enhance the reliability of coding. to enhance the trustworthiness of our findings, we will use a combination of duplicate coding by two researchers trained in both inductive analysis and the use of the tdf, peer debriefing activities and consensus-building measures. participants we will invite adult gbmsm (aged 18+) living in london (ontario) and calgary (alberta) to complete an anonymous questionnaire regarding their views about current and potential future barriers and enablers to donating source plasma. questionnaire items will enable the assessment of respondents' eligibility to donate according to the revised criteria but will not exclude participants to the survey on the basis of these criteria. recruitment and procedure our local advisory groups of gbmsm will facilitate recruitment by providing access to the venues and organisations through which a link to the online survey can be circulated as well as by advising on additional non-traditional venues for recruiting participants. we will offer the opportunity to enter a draw for one of ten pre-paid visa gift cards valued at cad$ 125 each. questionnaire development the questionnaire will be designed for completion by adult gbmsm and will include screening questions to ensure that we involve our targeted respondents. we will use items from a tdf questionnaire previously assessed for its discriminant content validity [32] to assess barriers and enablers to donating source plasma if they were eligible under revised eligibility cri-teria. appropriate language and response options will be informed by the interviews; thus, certain questions may be unique to each city. the questionnaire will be piloted by gbmsm prior to launching. data analysis our analytical approach is modelled on an approach used by presseau et al. [33] . we will conduct descriptive analyses to identify mean scores and standard deviations on each of the 14 tdf domains. as donation represents a hypothetical behaviour for respondents at the moment, we will assess intention to donate as a proxy for actual donation (consistent with other donation studies) [47] . we will assess bi-variate associations between intention to donate source plasma with socio-demographic factors, including self-identified gender, eligibility to donate (based on revised criteria) and responses to each tdf domain. informed by behavioural theory [33, 48] , we will then conduct multiple regression analysis to investigate which variables are associated with gbmsm's intention to donate source plasma. we will investigate whether these associations are moderated by their eligibility as determined by the revised criteria. if the surveys end up very similar between sites, we will analyse the combined sample and investigate whether the associations differ by location. we are powered to conduct independent analyses in each region if needed. sample size for analysis of our survey data, for a regression model comprised of data covering 20 independent variables [i.e. 13 tdf domains (not including intention), as well age, self-identified gender, rural/urban, sex with new partner in last 12 months (yes/no), sex with more than one partner in last 3 months (yes/no), in a monogamous relationship (yes/no), married (yes/no)], we will require a total sample size of 314 participants (157 participants in each region) to detect a medium effect size (r 2 = 0.15). we will aim to continue to recruit until this sample is exceeded or the recruitment period is completed (6 months). we will interview english-speaking donor centre staff involved in applying current eligibility criteria or discussing eligibility with potential donors at the london donor centre. recruitment will be facilitated by our stakeholder contacts at the donor centre. nurses and donor care associates will be invited to participate in a one-on-one semi-structured telephone interview lasting 30-45 min. interviews will focus on the barriers and enablers to using each of the proposed revised screening criteria. our approach to interview guide development, data analysis and sample size is rooted in the tdf framework and will follow the methods outlined for the interviews with gbmsm. the findings of the study may be of interest to diverse audiences and require a multi-pronged dissemination strategy. a key advantage of an ikt approach is that stakeholders are members of the research team and provide real-time guidance on the type of dissemination that is needed at different stages of the project. our first priority is to work with our stakeholders to ensure they have the findings in an appropriate format and to develop a dissemination plan for their respective communities. we will collaborate with our local advisory groups of gbmsm to disseminate among their local communities and beyond, which may involve a website and online events or articles in gbmsm-focused venues. we will also work with our blood operator stakeholders to disseminate appropriately through their organisations and international networks. beyond our stakeholders, dissemination of this work is likely relevant to stakeholders outside the traditional scientific audience and, thus, we will aim to publish in open-access journals or repositories and share these via social media to reach a broader audience. source plasma donation by sexually active gbmsm is not currently permitted. the data generated in this study is based on a hypothetical change in policy. the barriers and enablers will be elicited by asking respondents to answer to a hypothetical scenario, which may be different from their responses to real-life scenarios [49] . if revised eligibility is approved, ongoing investigation is needed alongside any initial implementation. this protocol is based on a grant funded by health canada, administered by canadian blood services, and peerreviewed by experts external to the funder and blood operator. the study has been reviewed and received ethics approval from the ottawa health science network research ethics board (id # 20190287-01h and 20200255-01h). the canadian blood services ethics board has also reviewed and approved the parts of the study involving donor centre staff (id 2019.020). at the time of submission, recruitment and data collection had begun with donor centre staff and gbmsm. the findings from this study will contribute to our understanding of the views of gbmsm regarding source plasma donation and source plasma donation eligibility policies as well as of the perceived barriers and enablers to donating source plasma should they become eligible. findings will help to inform the development of implementation strategies and support donation promotion if policies change to enable some sexually active gbmsm to donate. this study will also generate insights regarding staff anticipated barriers and enablers to implementing revised criteria for source plasma donation that will inform intervention development in preparation for wide-scale implementation if policy changes are approved by health canada. a strength of using the tdf is that the domains have been mapped to specific evidence-based behaviour change techniques [36] . thus, the findings will specify theory-driven and evidence-based strategies to be incorporated into future intervention development to address the barriers and enablers identified, increasing the likelihood that resulting strategies are likely to be effective. although the findings are based on two collection sites, by rooting our approach in behaviour change theory, we will enhance the transferability of findings to other sites. the multiple sources and types of data that will be collected in this study will enable a broader understanding of the needs to be addressed prior to implementation. qualitative methods will help us to explore the full range of factors that may impact implementation by staff and donation among gbmsm. quantitative findings will inform which of these factors identified by gbmsm are likely to have the greatest impact on donation intention, elucidating the areas most amenable to the targeted strategies to support donation. canada is not alone in its consideration of behaviourrisk screening for blood donation [50] [51] [52] . while many countries have followed a similar evolution to blood donation policies for gbmsm, the context in which these policies are made and enacted vary greatly. the contextual findings can help decision-makers and researchers in other jurisdictions to better situate the findings and assess the transferability of our findings to their context [42] . as of 2020, the public discourse related to blood donation by gbmsm was intensifying in response to the repeated calls for blood donation by blood operators internationally due to covid-19-related blood shortages [53, 54] . there was also increasing public interest in plasma donation and the ineligibility of gbmsm due to the clinical trials investigating convalescent plasma as a treatment for covid-19 [55, 56] . convalescent plasma is a type of plasma collected from patients recovered from covid-19; gbmsm who have recovered from covid-19 are not eligible to donate. the iterative and adaptable nature of an ikt approach that engages both the blood operator and gbmsm combined with the flexibility of qualitative methods makes this project uniquely situated to adapt and respond to the shifting social context of the study. due to the decades of exclusion from blood and bloodproduct donation that gbmsm have experienced and the social and political movement that has arisen in response, there may be divisions between blood operators in different countries and gbmsm that will need to be bridged for gbmsm to engage enthusiastically with a source plasma donation programme. research using ikt has the potential to create impact beyond addressing the research objectives. there is potential for the different knowledge user groups involved in the research to experience mutually beneficial learning cycles throughout the project's stages of design, data collection, analysis, reporting and dissemination [25] . as each unique perspective contributes to the work, all knowledge users and researchers learn from these perspectives and may shift their own perspectives. furthermore, in this process, there is the potential to strengthen relationships across knowledge users. partnerships between gbmsm and canadian blood operators can only aid in improving the development of appropriate and sensitive implementation supports and ultimately facilitate bringing gbmsm into the donor base. this research will explore the perceptions and experiences of gbmsm and donor centre staff regarding the feasibility and acceptability of implementing revised eligibility criteria for source plasma donation by gbmsm. the findings from this study will help refine or provide feedback on three proposed behaviour-based screening questions for donation eligibility and provide a basis for developing sensitive materials that could support a change in policy. abbreviations gbmsm: gay, bisexual, and other men who have sex with men; ikt: integrated knowledge translation; tdf: theoretical domains framework. donor criteria for men who have sex with men: a canadian perspective state ment-from-the-minis ter-of-healt h-on-furth er-reduc ing-barri ers-for-blood -donat ion-by-men-who-have-sex-with-men donor deferral policies for men who have sex with men: past, present and future sexual risk behaviour and donor deferral in europe changing blood donor screening criteria from permanent deferral for men who have sex with men to individual sexual risk assessment: no evidence of a significant impact on the human immunodeficiency virus epidemic in italy the evolving blood donor deferral policy for men who have sex with men: impact on the risk of hiv transmission by transfusion in france one step closer": acceptability of a programme of plasma donation for fractionation from men who have sex with men views and experiences of men who have sex with men on the ban on blood donation: a cross sectional survey with qualitative interviews saving lives, maintaining safety, and science-based policy: qualitative interview findings from the blood donation rules opinion study (blood drops) context and social perceptions of blood donation in donors found positive for human immunodeficiency virus in france should men who have ever had sex with men be allowed to give blood? no is there a right to donate blood? patient rights; donor responsibilities should men who have ever had sex with men be allowed to give blood? yes end the gay blood ban. the guardian blood donation, deferral, and discrimination: fda donor deferral policy for men who have sex with men reconsidering the lifetime deferral of blood donation by men who have sex with men negotiating exclusion: msm, identity, and blood policy in the age of aids the paradoxical situation of blood donation in the haitian-quebec community transition to a 1-year deferral for male blood donors who report sexual contact with men: staff perspectives at one blood collection organization what can qualitative research do for randomised controlled trials? a systematic mapping review developing and evaluating complex interventions: the new medical research council guidance how do people become plasma and platelet donors in a vnr context? developing theory-informed behaviour change interventions to implement evidence into practice: a systematic approach using the theoretical domains framework knowledge translation in health care. hoboken: wiley embracing complexity and uncertainty to create impact: exploring the processes and transformative potential of co-produced research through development of a social impact model how does integrated knowledge translation (ikt) compare to other collaborative research approaches to generating and translating knowledge? learning from experts in the field building an integrated knowledge translation (ikt) evidence base: colloquium proceedings and research direction lost in knowledge translation: time for a map? making psychological theory useful for implementing evidence based practice: a consensus approach. qual saf health care validation of the theoretical domains framework for use in behaviour change and implementation research a guide to using the theoretical domains framework of behaviour change to investigate implementation problems discriminant content validity of a theoretical domains framework questionnaire for use in implementation research identifying determinants of medication adherence following myocardial infarction using the theoretical domains framework and the health action process approach • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year submit your research ? choose bmc potential determinants of health-care professionals' use of survivorship care plans: a qualitative study using the theoretical domains framework from theory to intervention: mapping theoretically derived behavioural determinants to behaviour change techniques use of serial qualitative interviews to understand patients' evolving experiences and needs qualitative interviews in medical research the qualitative research interview methods of data collection in qualitative research: interviews and focus groups determining sample size naturalistic inquiry. thousand oaks: sage; 1985 sample size in qualitative interview studies: guided by information power characterising and justifying sample size sufficiency in interview-based studies: systematic analysis of qualitative health research over a 15-year period using thematic analysis in psychology three approaches to qualitative content analysis factors explaining the intention to give blood among the general population modeling health behavior change: how to predict and modify the adoption and maintenance of health behaviors what we say and what we do: the relationship between real and hypothetical moral choices an end to lifetime blood donation ban in israel for msm would be a major step toward a science-based policy that reduces stigma blood donation deferral policies among men who have sex with men in brazil men having sex with men and blood donation: is there a game changer on the horizon? relaxes rules for gay blood donors amid coronavirus-will canada go further? global news pm trudeau urges canadians to donate blood during covid-19 pandemic i recovered from covid-19. but i can't donate my plasma because i'm gay publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. jp and mg conceived the study. all authors participated in the design of the study. jp and ev led the drafting of the manuscript with input from all authors. all authors read and approved the final manuscript. this study received funding from canadian blood services msm research grant program and canadian blood services msm plasma program, funded by the federal government (health canada) and the provincial and territorial ministries of health. the views herein do not necessarily reflect the views of canadian blood services or the federal, provincial, or territorial governments of canada. not applicable. key: cord-326176-n0xo3e53 authors: patel, reema t.; caceres, ana; french, adrienne f.; mcmanus, patricia m. title: multiple myeloma in 16 cats: a retrospective study date: 2008-03-05 journal: vet clin pathol doi: 10.1111/j.1939-165x.2005.tb00059.x sha: doc_id: 326176 cord_uid: n0xo3e53 background: there is limited published information regarding feline multiple myeloma. diagnostic criteria are derived from canine studies and to our knowledge, have not been critically reviewed for cats. objective: to evaluate the clinical and laboratory findings in cats with multiple myeloma and appraise diagnostic criteria. methods: retrospective evaluation of medical records was performed. inclusion required an antemortem diagnosis of multiple myeloma using 2 of 4 criteria: 1) ≥20% plasma cells in the bone marrow, or ≥10% if atypical plasma cells; 2) paraproteinemia; 3) radiographically‐evident osteolysis; 4) light chain proteinuria. alternatively, a postmortem diagnosis was based on the findings of multiple plasma cell neoplasms, with marrow involvement. results: sixteen cats were diagnosed with multiple myeloma between 1996 and 2004, with a median age of 14.0 years; 9 of 16 (56%) were castrated males, and 7 of 16 (44%) were spayed females. laboratory abnormalities included hyperglobulinemia (14/16, 87.5%), with 11/14 (78.5%) monoclonal and 3/14 (21.4%) biclonal gammopathies; hypoalbuminemia (4/16, 25%); light chain proteinuria, (4/9, 44.4%); hypocholesterolemia (11/16, 68.7%); hypercalcemia, (3/15, 20%); nonregenerative anemia, (11/16, 68.7%); regenerative anemia, (1/16, 6.2%); neutropenia (5/15, 33.3%); thrombocytopenia (8/16, 50%); and marrow plasmacytosis (14/15, 93.3%). plasma cells were markedly immature, atypical, or both in 10 of 12 (83.3%) cats. focal or multifocal osteolysis was noted in 6 of 12 (50%) cats for which radiographs were available for review; generalized osteopenia was found in 1 (8.3%) cat. noncutaneous, extramedullary tumors were found in all cats assessed, 7/7 (100%), including spleen (6), liver (3), and lymph nodes (4). the disease in 1 of 2 cats with cutaneous tumors progressed to plasmacytic leukemia. conclusions: common findings in feline multiple myeloma include atypical plasma cell morphology, hypocholesterolemia, anemia, bone lesions, and multi‐organ involvement. based on the results of this study, we advocate modifying diagnostic criteria in cats to include consideration of plasma cell morphology and visceral organ infiltration. multiple myeloma is a multifocal plasma cell neoplasm involving bone marrow. multiple myeloma patients present with nonspecific and insidious clinical signs such as lethargy, inappetence, weight loss, and intermittent vomiting. 1 it is a rare neoplasm in cats, with an estimated incidence of ,1% of all feline hematopoietic neoplasms. 2, 3 there are few reports in the literature, with most of these limited to 1 or 2 case studies [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] the largest retrospective report to date was limited to 4 cats. 1 in addition, authors of a review article on causes of monoclonal gammopathy in dogs and cats included limited information regarding 6 cats diagnosed with multiple myeloma over a 6-year period at the animal medical center in new york city. 15 most information regarding the biology of this disease is derived from human and canine studies. additional information regarding behavior of the disease in cats and applicability of currently used diagnostic criteria in cats is needed. therefore, we conducted a retrospective study using biopsy, cytology, and necropsy files from the matthew j. ryan veterinary hospital of the university of pennsylvania (mjr-vhup) school of veterinary medicine. we reviewed signalment, clinical features, clinicopathologic findings, and outcome. we compared features seen in cats with multiple myeloma to those historically noted in dogs and people. cats diagnosed with multiple myeloma from january 1, 1996 to february 1, 2004 were identified via a computer search of biopsy, necropsy, and cytology reports, and coded discharge diagnoses in the mjr-vhup database. inclusion in the study required 2 of 4 antemortem criteria: 1) bone marrow plasmacytosis with !20% plasma cells, or !10% atypical plasma cells; 2) monoclonal or biclonal gammopathy on serum protein electrophoresis (spe); 3) radiographic evidence of osteolysis; and 4) light chain (bence-jones) proteinuria. a plasma cell population was defined as morphologically atypical if .5% of the plasma cells had morphology not associated with normal reactive plasmacytosis, eg, marked anisocytosis, marked anisokaryosis, multinucleation with .2 nuclei, immature chromatin, distinct nucleoli, maturation asynchrony, clefted nuclei, and markedly variable n:c ratios. [16] [17] [18] necropsy diagnoses were based on finding plasma cell tumors with multiple organ and marrow involvement. incidence of multiple myeloma was calculated by dividing the number of cats with multiple myeloma by the total number of cats with malignancies and by the total number of cats with hematopoietic neoplasms. in bone marrow aspirate smears stained with wright-giemsa (harleco, gibbstown, nj) the percentage of plasma cells was determined by assessing 400-500 nucleated cells at the time of the original diagnosis; percentages were rechecked retrospectively by one of the authors (rtp) for the purpose of this study. histologic preparations were stained with h&e. when histologic sections were used in lieu of aspirates, ''plasma cell mass'' as a percentage of marrow volume was assessed retrospectively (instead of plasma cell percentage) for this study by one of the authors (aff). 17 complete blood counts were run on an abbott cell-dyn 3500 hematology analyzer (plato, texas, usa) with multi-species settings. all differential leukocyte counts were determined manually by counting 100 cells and all blood smears also were reviewed by one of the authors (rtp). routine serum chemistry profiles were analyzed using an ortho-clinical diagnostics vitros 250 (rochester, new york, usa). ionized calcium concentration was determined in some of the cats using a nova stat profile m (waltham, massachusetts, usa). proteinuria was detected using bayer multistix 10sg reagent strips (elkhart, indiana, usa) read on a clinitek 50 urine chemistry analyzer (bayer). all urine samples were also screened for protein by sulfosalicylic acid (ssa) precipitation. if positive by ssa, urine samples were then tested for light chain proteinuria by either the bence-jones heat precipitation method 19 or by immunoelectrophoresis. light chain proteinuria was determined by the heat precipitation method in 7 cats and by urine protein electrophoresis in 2 cats. protein electrophoresis using capillary gel electrophoresis was done on serum from 14 cats (paragon electrophoresis system, beckman coulter inc, ramsey, mn, usa) to evaluate the presence of a monoclonal protein spike (m-protein or mspike). the stained cellulose acetate strip was scanned with an appraise clinical densitometer (beckman). immunoelectro-phoresis was done (helena laboratories, beaumont, texas, usa) using species-specific anti-immunoglobulin (ig) g, anti-igm, and anti-iga to identify the specific type of m-protein in 2 cats. serum viscosity, relative to water, was determined in 1 cat using a wells-brookfield cone-plate viscometer (oakville, ontario, canada), with a ratio .3.5 interpreted as abnormal. 6 eight of the 16 cats had feline coronavirus antibody titers determined via serum elisa (antech diagnostics, lake success, ny, usa). all cats were tested for feline leukemia virus (felv) and feline immunodeficiency virus (fiv) using the snap combined immunoassay for felv antigen and fiv antibody (idexx laboratories, westbrook, me, usa). sixteen cats met the diagnostic criteria; 13 were patients at mjr-vhup and 3 had samples submitted through the external biopsy service. patient age ranged from 7 to 18 years, with a median of 14 years and mean of 12.6 years. there were 9 castrated males (56%) and 7 spayed females (44%); 13 were domestic shorthair, 2 were domestic longhair, and 1 was a russian blue cat. the incidence of multiple myeloma at mjr-vhup during the study period was 0.9% of all cats diagnosed with malignancies (n51491) and 1.9% of all cats diagnosed with hematopoietic neoplasms (n5670), including lymphoma, during the same period. historical and physical examination abnormalities were nonspecific (table 1) , with the exception of 3 cats. cat 5 had multiple skin masses on the xiphoid, right hock, left flank, cranial thorax, and lumbar back and a gallop cardiac rhythm. cat 8 had a multilobulated cutaneous tarsal plasma cell tumor diagnosed histologically 6 months previously. diagnostic evaluation (marrow, serum protein electrophoresis, radiographs) at the time of initial presentation for the tarsal mass was negative for systemic disease or local bone involvement. cat 10 had an m-protein first detected 9 years earlier. a diagnosis of monoclonal gammopathy of undetermined significance (mgus) was made based on lack of evidence for multiple myeloma; the cat had no clinical signs, no marrow plasmacytosis, no significant radiographic or ultrasonographic findings, and negative light chain proteinuria. relevant laboratory results and bone marrow and extramedullary tissue involvement were summarized (tables 2-4 ). cat 5, with only mild hyperglobulinemia, had hyperviscous serum, based on viscometry. immunoelectrophoresis for cats 8 and 13 identified the m-protein as iga and igg, respectively. four cats (nos. 2, 7, 9, and 12) were hypoalbuminemic, with albumin values ranging from 1.8-2.3 g/dl (reference interval 2.4-3.8 g/dl). protein:creatinine ratios (p:c) were determined in 2 cats with proteinuria. cat 1 had 2þ proteinuria and a p:c of 0.62 (reference interval 0.5-1.0). cat 15 had 3þ proteinuria and a p:c of 19.4. light chain proteinuria, detected by urine protein electrophoresis, was found in both cats tested (nos. 3 and 6). a monoclonal spike was observed in the beta region in cat 3 and in the gamma region in cat 6. four cats (nos. 1, 3, 11, and 16) were diagnosed with renal failure based on increased urea (32-56 mg/dl, reference interval 15-29 mg/dl) and creatinine (2.1-3.4 mg/dl, reference interval 0.5-2.0 mg/dl) concentrations, accompanied by inadequately concentrated urine in the presence of dehydration. one cat (no. 2) had increased creatinine concentration (2.9 mg/dl), a urea concentration within the reference interval (29 mg/dl), and a urine specific gravity of 1.024, despite clinical dehydration. seven of 14 cats had aspartate aminotransferase activities .2 times the upper limit of the reference interval, with results ranging from 82 to 420 u/l (reference interval 1-37 u/l). cat 6 had concurrent increases in alanine aminotransferase (222 u/l, reference interval 33-152 u/l) and alkaline phosphatase (244 u/l, reference interval 22-87 u/l) activities, and hyperbilirubinemia (2.2 mg/dl, reference interval 0.1-0.8 mg/dl). three of 15 cats tested had hypercalcemia at initial presentation. cats 1 and 16 had total calcium concentrations of 14.6 and 13.5 mg/dl (reference interval 9.1-11.2 mg/dl) and ionized calcium concentrations of 1.61 and 1.68 mmol/l (reference interval 1.12-1.40 mmol/l), respectively. cat 9 had a total calcium concentration of 12.9 mg/dl; ionized calcium was not determined. hyperphosphatemia was found in 3 cats (nos. 7, 8, and 11), with phosphorus values ranging from 6.1-8.8 mg/dl (reference interval 2.5-6.0 mg/dl). twelve of 16 cats had mild to severe anemia (table 2 ), all but one of which were nonregenerative, normocytic, and normochromic. cat 7 had severe regenerative, macrocytic hypochromic anemia, with 2þ polychromasia; however, the cause of the anemia was not determined. intratubular pigment casts, interpreted as presumptive evidence of hemoglobinuria, were seen in tissue sections taken at necropsy, so intravascular hemolysis was suspected. five cats were neutropenic ( table 2) . one cat (no. 5) had mild leukocytosis due to mild neutrophilia, concurrent with myeloid hyperplasia in the marrow aspirate. cat 4, with a neutrophil count within the reference interval, had mild toxic change in its neutrophils. neutrophil morphology was unremarkable in all remaining cats and there was no antemortem evidence of infection, other than periodontal disease, which was clinically assessed as incidental. plasma cells were detected in peripheral blood from 4 cats. cat 8 had a leukocyte count within the reference interval but had 53% (3,000 cells/ll) plasmablasts and plasma cells ( figure 1 ). cat 9 was both leukopenic and neutropenic, and had occasional moderately immature lymphocytes and welldifferentiated plasma cells (,1% of total leukocytes) in peripheral blood. cats 12 and 16 had total leukocyte and neutrophil counts within reference intervals and rare mature plasma cells. cat 4 was presented for intermittent epistaxis, but platelets were judged to be adequate on smear evaluation. in 2 cats in which prothrombin time and partial thromboplastin none of the 8 cats tested for antibody to feline coronavirus had positive titers and no cats were positive for felv or fiv infections. thoracic and abdominal radiographs were available for review by one of the authors (ac) for 12 of the 14 cats on which radiography was performed. eight cats (67%) had cardiomegaly; of these, 2 had pulmonary vascular enlargement and cardiogenic pulmonary edema. hepatomegaly was seen in 7 (58%) cats and splenomegaly was seen in 4 (33%). in cat 2, renomegaly was seen in addition to hepatosplenomegaly and cardiomegaly. cat 9 had poor peritoneal detail secondary to peritoneal effusion, which impaired evaluation of abdominal organs. radiographically, skeletal abnormalities were observed in 7 of the 12 cats evaluated. cat 1 had multiple punctate lytic lesions in the femur, tibia, pelvis, and lumbar vertebrae. cat 3 had diffuse osteopenia, based on the sclerotic appearance of the lumbar vertebral endplates and a thin double cortical line of the scapular spine. a single punctate lytic lesion was observed in the distal tibia in cat 5, in the proximal humerus in cat 7, and in the femur in cat 11. cat 14 had a locally invasive aggressive lytic lesion in the left olecranon extending into the proximal ulnar metaphysis ( figure 2 ). cat 16 had multiple small lytic lesions in the proximal humerus and cranial lum-bar vertebrae and an expansile lesion in a rib resembling an active callus. ultrasound was performed in 7 cats (nos 2-7 and 9), all of which had abnormalities in the spleen. these included splenomegaly and mottled spleen (5/7), multiple hypoechoic nodules (3/7), multiple hyperechoic nodules (1/7), and diffusely hypoechoic spleen (2/7). five of the 7 cats (71%) examined by ultrasound had 1 or more hepatic abnormalities, including hepatomegaly (3/7), diffusely hyperechoic livers (4/7); and a 7-mm in diameter hyperechoic nodule on the right lateral liver lobe and distended hepatic veins (1/7). the latter cat also had cardiomegaly and enlarged pulmonary vessels. diffusely hyperechoic, enlarged kidneys were seen in 1 cat and enlarged hypoechoic medial iliac lymph nodes were seen in another cat. marrow plasmacytosis (13 to .99% plasma cells) was detected in bone marrow aspirates (11/15) or core biopsies (3/15) in 14 of the 15 cats from which diagnostic samples were obtained (table 3) . plasma cell morphology in aspirate preparations ranged from well differentiated (2/12, 16.7%) ( figure 3 ) to atypical (10/12, 83.3%) (figure 4 ). atypical features included increased cell size, multiple nuclei, clefted nuclei, moderate to marked anisocytosis and anisokaryosis, variable n:c ratios, decreased chromatin density, and variably distinct, variably sized nucleoli. plasma cells in several cats had ''flame cell'' morphology (table 3) , characterized by peripheral ydiagnosis of multiple myeloma made at necropsy. zorgan/site involvement determined histologically; all other sites determined cytologically. pink-stained cytoplasmic processes ( figure 5 ). in 1 cat in which multiple myeloma was diagnosed by biopsy of a lytic lesion in the left olecranon (no. 14), relatively welldifferentiated plasma cells comprised almost 100% of bone marrow mass ( figure 6 ). cat 11 lacked demonstrable marrow plasmacytosis, but the aspirate was taken from the radiographically unremarkable humerus, whereas the lytic lesions were detected in the pelvis and femur. aspiration of the lytic lesions was not attempted. bone marrow m:e ratios were decreased in 3/11 cats and increased in 4/11. in addition to plasmacytosis, marrow changes included hemosiderosis (cats 3, 5, 9, and 10), myeloid hypoplasia (cats 1 and 10), erythroid hypoplasia (cats 5, 10, and 15), ineffective erythropoiesis (cats 2, 4, 12, and 16), ineffective erythroid hyperplasia (cat 3), myeloid hyperplasia (cat 5), and ineffective hematopoiesis (cat 13). noncutaneous, extramedullary tumors were detected in all 7 cats where this was assessed. sites included spleen, liver, and lymph nodes ( table 3 ). the lymph nodes were mesenteric in 3 cats and both mesenteric and iliac in the cat with the single, tarsal cutaneous plasma cell tumor. plasma cell infiltrates did not always correlate with splenomegaly, hepatomegaly, or lymphadenopathy. if enlarged organs were aspirated, they were invariably infiltrated, but myeloma cell infiltrates were also detected in organs of normal size. five cats with ultrasonographic changes in the spleen had either histologic (1 cat at necropsy) or cytologic (4 cats) evidence of tumor involvement. the cutaneous masses noted in cat 5 were diagnosed as plasma cell tumors by cytology. one cat (no. 9) presented with pleural and peritoneal effusions, all of which were characterized as modified transudates, with no neoplastic cells or infectious agents noted on cytologic examination. necropsy findings confirmed the diagnosis of multiple myeloma in cats 7 and 9. gross lesions in cat 7 included moderate icterus, hepatosplenomegaly, cardiomegaly, and generalized lymphadenopathy. on histopathology, marked plasmacytosis was noted in bone marrow, spleen, liver, and mandibular, retropharyngeal, prescapular, and axillary lymph nodes. plasma cells were typical to atypical with anisocytosis, anisokaryosis, decreased n:c ratios, and occasionally, binucleation. moderate plasmacytosis also was noted in periportal areas and sinusoids within the liver. the bone marrow was hypercellular (90-95% cellularity) with typical and atypical plasma cells distributed unevenly among normal marrow elements; plasma cell mass comprised approximately 50% of bone marrow mass. cat 9 was emaciated, with moderate fibrinous peritoneal effusion and severe serosanguinous pleural effusion. superficial, mesenteric, bilateral iliac, and cranial mediastinal lymph nodes were moderately to severely enlarged, and hepatosplenomegaly and cardiomegaly were noted. marked infiltration of typical and atypical plasma cells was detected on histopathologic sections of bone marrow, spleen, and lymph nodes. atypical cells were similar to those described in case 7. in the bone marrow, plasma cells were (c) the plasma cell on the left has a small, slightly oval, eccentric nucleus, moderately dense chromatin, and scant cytoplasm with fine, wispy cytoplasmic processes. the cell on the right is larger and has a higher n:c ratio, consistent with a plasmacytoid lymphoblast or plasmablast, but chromatin is atypically dense and granular (maturation asynchrony). wright's-giemsa, bar57.5 lm. multiple myeloma is an uncommonly diagnosed disease in cats and has been reported infrequently in the literature. the cats in this report bring the total number of reported feline multiple myeloma cases to 47. 1,315 we determined the frequency of multiple myeloma in cats at mjr-vhup to be slightly ,1% of all malignant neoplasms in cats during the same time period. canine multiple myeloma during the same time period at mjr-vhup was diagnosed at a much lower frequency, accounting for only 0.3% of all malignancies in dogs (mcmanus p, unpublished data). absolute numbers of canine cases outnumbered feline cases during these 8 years, but only because our hospital sees a substantially higher number of canine patients. multiple myeloma accounted for approximately 2% of all hematopoietic neoplasms in both dogs (mcmanus p, unpublished data) and cats at our hospital. this incidence was higher than that previously reported, but still is much less than the rate in humans, where multiple myeloma is the second most common hematopoietic neoplasm, accounting for 13% (caucasians) to 33% (african americans) of all hematopoietic tumors. 20 multiple myeloma is extremely rare in other domestic species with only a few reported cases in horses. [21] [22] [23] [24] [25] [26] [27] [28] [29] multiple myeloma is generally a disease of older animals. only 4 cats in our study were ,10 years old, and the average age was 12.5 years. in people, the average age at diagnosis is 66 years. 30 in our study of 16 cats, males accounted for 56% of cases. pooling information from previous reports, males account for 63.8% (30/47) of all feline multiple myeloma cases. this sex predisposition is similar to what occurs in people, where the largest retrospective study, of 1027 patients, included 59% men. 30 there is no apparent gender predilection in dogs; the largest retrospective study to date (60 dogs) included 30 males and 30 females. 31 as in other species, multiple myeloma is not likely to be directly caused by a specific viral infection. a link between human immunodeficiency virus and progression to multiple myeloma has not been established, 32, 33 and cats in this study were negative for fiv and felv infection. the results of this study reinforced the impression that clinical signs of feline multiple myeloma, as in other species, are usually nonspecific, eg, lethargy, vomiting, renal failure, hemostatic abnormalities, anorexia, and diarrhea. the first veterinary clinical pathology clue to a diagnosis is usually hyperglobulinemia, which was found in 87.7% of animals in this study. current published recommendations for determining a diagnosis in animals initially appear straightforward, in that 2 of the following 4 criteria are required: 1) bone marrow plasmacytosis with .20% plasma cells, 2) monoclonal gammopathy based on spe, 3) osteolysis, and 4) light chain (bence-jones) proteinuria. 2 these criteria are unweighted for animal patients; in people, criteria are weighted as ''major'' and ''minor'' and accommodate lower plasma cell percentages. 17 in people, confirmation of myeloma requires first that the patient be symptomatic (ie, have bone pain) or have anemia, hypercalcemia, azotemia, hypoalbuminemia, or bone demineralization. the diagnostic criteria of multiple myeloma are then applied. major criteria include 1) plasmacytoma(s) with biopsy, 2) marrow plasmacytosis .30%, 3) m-protein with .3.5 g/dl igg or .2.0 g/dl iga, and 4) kappa or lambda light chain excretion .1.0 g/dl on 24-hour urine protein electrophoresis. the 4 minor criteria include a) marrow plasmacytosis with 10-30% plasma cells, b) m-protein at values less than indicated above, c) lytic bone lesions, and d) ,50% normal serum ig concentration. if the diagnosis includes major criteria, then any 2 of the 4 will suffice, or major criterion 1 plus minor criterion b, c, or d; or major criterion 3 plus minor criterion a or c. if the diagnosis is based on only minor criteria, then it must include the first and second criteria (a and b), plus 1 of the remaining 2 criteria (c or d). in our study we didn't imitate this system, but we did modify the requirements for animals by including plasma cell atypia as a criterion when marrow plasmacytosis was between 10 and 20%, which occurred in 3 cases. in humans, nuclearcytoplasmic maturation asynchrony, nuclear immaturity, and pleomorphism are considered reliable markers for distinguishing neoplastic cells from reactive plasma cells. 16, 17 in addition, reactive plasma cells usually do not exceed 5% of all nucleated cells in marrow and are well differentiated. [16] [17] [18] although we required a minimum of 10% marrow plasmacytosis for diagnosis, it should be noted that even the 10% threshold can be problematic because enumeration of plasma cells in routinely stained bone marrow aspirate smears may significantly underestimate plasmacytosis when compared to immunohistologic examination. 34 in a study of 176 human patients with multiple myeloma, where diagnosis was based on immunohistologic techniques, 40% of patients had ,10% plasma cells in bone marrow aspirates. 34 using the modified guidelines described here, the most commonly paired criteria in cats were marrow plasmacytosis and paraproteinemia, which were found in 12 of 16 cats. of the remaining 4 cats, 1 lacked a marrow aspirate and 2 lacked spe. only 1 cat lacked demonstrable marrow involvement, and the diagnosis was based on lytic lesions and a monoclonal spike. results of bone marrow evaluation were normal, but the aspirate had been taken some distance away from the lytic lesions. this negative marrow result likely reflected the focal nature of multiple myeloma and served to emphasize the importance of a core biopsy or multiple marrow aspirates from different sites, particularly affected bone sites. although we did not use extramedullary involvement as a diagnostic criterion, it was a common finding in the cats in this study, suggesting that assessment of extramedullary sites could assist in the diagnostic evaluation of hyperglobulinemic cats, especially when marrow aspirates cannot be obtained. this is in contrast to dogs, where extramedullary involvement is a less consistent finding (van winkle t, unpublished observations based on mjr-vhup necropsy reports for canine multiple myeloma). using the 4 criteria as currently defined in veterinary medicine could potentially result in a diagnosis of multiple myeloma based only on the presence of an m-protein and light chain proteinuria, raising the question as to whether these criteria are sufficient. in this study, 1 cat met only these 2 criteria; however, bone marrow aspirates were not done because both liver and spleen were infiltrated with plasma cells. in such cases, perhaps an m-protein and light chain proteinuria should be considered together as constituting 1 criterion rather than separate entities, and, as discussed previously, plasma cell atypia and extramedullary involvement should be included to assist in diagnosis. the presence of a monoclonal protein (m-protein or m-spike) reflecting production of a single ig is one of the most discriminating clues that a plasmacytosis is neoplastic, ie, clonal, as opposed to reactive. in people, an m-protein is found in the serum or urine of .97% of myeloma patients, 30 with remaining cases classed as nonsecretory. in the 14 cases in which spe was performed in our study, 11 (78.5%) had monoclonal proteins, and 3 had biclonal spikes (21.4%), all migrating in the gamma region. possible causes for paired spikes include 2 independent secretory tumor cell lines, 35 the divergence of the primary tumor into 2 separate plasma cell populations producing distinct m-proteins, 1 clone of myeloma cells producing 2 distinct paraproteins, or spurious biclonal peaks due to split dimeric or multimeric paraproteins, eg, iga. 4 multiple myeloma is the most common cause of mproteins; however, other conditions occasionally can induce a monoclonal gammopathy in small animals, such as chronic infection (leishmaniasis, 36,37 ehrlichiosis, 37-40 chronic pyoderma, 41 feline infectious peritonitis), amyloidosis, b-cell lymphoma, 37 waldenströ ms macroglobulinemia, 37, 42 and mgus. 43 our study included a cat with a history of monoclonal gammopathy but no abnormal physical, hematologic, chemical, or radiographic findings at the time of first detection. this cat met all criteria for a diagnosis of mgus, which includes m-protein and ,10% bone marrow plasmacytosis, with no evidence of lytic lesions, light chain proteinuria, or other clinical, hematologic, and biochemical abnormalities. 44, 45 mgus occurs in 1-2% of people over the age of 50 and in 3% of people over the age of 70. 44,45 a significant proportion (25%) of these will evolve within 20 years into multiple myeloma, primary amyloidosis, macroglobulinemia, or another lymphoproliferative disease. 45 to the authors' knowledge, this is the first reported case of mgus in a cat. this cat first developed mgus as a young adult of 4 years old, and it took almost half of the cat's lifetime (9 years) to progress to multiple myeloma. diagnostic criteria in veterinary medicine do not currently hinge on determination of the type of ig; therefore, clinicians in our hospital rarely request this test. only 2 of the cats in this study (1 with iga and 1 with igg) had ig type determined. of the 25 published feline myeloma cases with immunoelectrophoresis results, including the 2 cases above, 20 had igg gammopathies, and 5 had iga gammopathies. 1, 3, 4, 6 common manifestations of bone lesions in multiple myeloma in both people and animals include classic punched-out lytic areas, generalized osteopenia, and pathologic fractures. 30 in this study, the percentage of cats with radiographically-evident skeletal lesions was 58.3%, which was higher than the 20% previously described for cats, 3, 4, 13, 15, 46 but similar to the 50-56% occurrence reported for dogs. 15, 31 the reason for the difference in our study compared to previous studies in cats may be increased clinician awareness of the lesion, better quality radiographs, and increased knowledge about the disease. the focal lytic lesions reported in cats affect pelvis, ribs, vertebral columns, and long bones, whereas humans also have skull lesions. 17 skull films were not done for any of the 16 cats. other causes of focal osteolysis are rare, and include carcinomas, 47 giant cell tumors of bone, 48 benign aneurysmal bone cysts, [49] [50] [51] and bone lesions secondary to tumor invasion. [51] [52] [53] one cat had generalized osteopenia, which was diagnosed radiographically. demineralization of bone in humans is detected through measurement of bone mineral density, a technique not used routinely in the assessment of animals. generalized osteopenia is not specific for multiple myeloma and may be seen with nutritional, renal, and metabolic disorders. [54] [55] [56] [57] multiple myeloma-associated hypercalcemia is not reported as frequently as bone lysis, 15, 30, 31 perhaps because disease progression is usually slow, allowing for appropriate metabolic controls. in our study, 20% of cats were hypercalcemic based on total calcium concentration. an ionized calcium test is needed to confirm hypercalcemia, because binding of calcium by the m-protein will increase total calcium concentration while ionized calcium remains within normal limits. 13 increased ionized calcium concentration, as seen in 2 of the cats in this study, supports true hypercalcemia. paraneoplastic hypercalcemia can result from lysis of bone either by tumor expansion or by osteoclast activation. 13 a recent retrospective study of 71 cases of hypercalcemia in cats over a 6-year period found that neoplasia (primarily lymphoma and squamous cell carcinoma), renal failure, and urolithiasis were the most commonly seen causes of increased total calcium concentration. none of the cats with neoplasia was diagnosed with multiple myeloma. 58 this likely reflects the low prevalence of this disease, rather than the frequency of hypercalcemia in myeloma patients, which, given the results of our study, is probably higher for myeloma than for any other feline neoplasm. light chain proteinuria was noted in 44.4% of cats in our study. taking into account these 9 cases plus previously reported cases, light chain proteinuria is found in 64.7% of feline multiple myeloma patients. 1, 3, 4, 59 screening for light chain proteinuria should not be limited to the urine dipstick method for proteinuria, because dipsticks primarily detect albuminuria. the ssa test is more sensitive to globulins and thus can be positive when the dipstick is negative, but it is nonspecific in that it detects albumin, globulins, bence-jones protein, polypeptides, and proteases. 60 false-positive ssa results may occur with penicillin and its derivatives, tolbutamide or sulfisoxazole metabolites, or certain roentgenographic contrast media in the urine. 60 in people, it has been reported that false-positive results for bence-jones proteins, assayed by heat precipitation, may be due to an excess of polyclonal light chains in patients with connective tissue diseases, chronic renal failure, or nonplasmacytic malignancies. 61 for these reasons, protein electrophoresis of concentrated urine is the preferred technique for detection of monoclonal light chains in urine. 2, 61 there are limited reports on the radiographic and ultrasonographic findings of internal organs in feline multiple myeloma. radiographic abnormalities in this study included hepatomegaly (58.3%), splenomegaly (25.0%), cardiomegaly (66.6%), and renomegaly (9.0%). the differential diagnoses for each individual finding is extensive and rarely includes multiple myeloma, but for hepatomegaly, splenomegaly, and cardiomegaly in combination with history and blood test results, the list is shorter. common neoplastic conditions that can produce this triad of organomegaly in cats include lymphoma and mast cell tumors. in this study we demonstrated that feline multiple myeloma, although not common, also induces these radiographic changes. cardiomegaly and cardiac disease, if present, may be explained by excessive cardiac workload and myocardial hypoxia secondary to hyperviscosity, as previously described in people. 62 the most common ultrasonographic abnormalities involved the spleen and liver and to a lesser extent the kidneys. the most consistent finding was splenic enlargement and diffuse or nodular hypoechogenicity. aspiration cytology or biopsy of ultrasound-detected splenic lesions was always diagnostic for plasma cell infiltration. even though a small number of cats was evaluated in this study, these ultrasonographic changes may be an important new finding that contributes to fast and accurate diagnoses of plasma cell neoplasms because of the ease and low morbidity and mortality from fine needle aspiration of spleen. although not a common disease in cats, plasma cell myeloma should be added to the list of differential diagnoses for an enlarged, mottled, hypoechoic spleen. the most consistent hepatic abnormality was diffuse hyperechogenicity and enlargement. the most common causes for a diffusely hyperechoic liver in cats are lipidosis and lymphoproliferative disease. hepatic lipidosis could not be ruled out as a contributing factor in most of the cats in this study, especially with the history of anorexia and lethargy; however, 1 cat with hyperechoic liver had diffuse infiltration of malignant plasma cells on necropsy and no evidence of hepatic lipidosis. hypocholesterolemia affected 68.7% of cats in this study, far more than the 26% reported for human patients. 30 serum cholesterol concentration is thought to correlate inversely with globulins concentration. 30 cat 4, for example, had one of the highest globulin concentrations (12.5 g/dl) and one of the lowest cholesterol concentrations (45 mg/dl). one postulated explanation is down-regulation of cholesterol production by the liver to maintain oncotic pressure in the face of hyperglobulinemia. 63 no cats had evidence of protein-losing enteropathy or severe malnutrition as a cause for decreased cholesterol, and only cat 6 had evidence of hepatic functional insufficiency. hypocholesterolemia can be seen with hyperthyroidism; however, none of the cats in this study were tested for total or free t4 concentration. complications secondary to hyperglobulinemia include hyperviscosity syndrome (hvs) and coagulation defects. typical signs of hyperviscosity include seizures, congestive heart failure, and retinal hemorrhages. 6 iga and igm are more often associated with hvs, because iga can dimerize or polymerize and igm is a pentamer. hvs has been reported previously in 4 of 11 cats in which retinal hemorrhages, neurologic signs, or both were noted. 6, 8 three had igg gammopathies, and 1 had iga gammopathy. one cat in this study had hyperviscous serum; however, immunoelectrophoresis was not done to determine the type of ig. cat 7 had bilateral retinal hemorrhages and a prolonged ptt, signs consistent with hyperviscous serum; however, serum viscosity was not measured. coagulation defects can result from paraprotein interference with clotting factors, protein coating of platelets leading to thrombocytopathy, and binding of the fab fragment of the m-protein to fibrin, preventing aggregation. 64, 65 only 1 cat in this study was suspected clinically of having coagulopathy (epistaxis), but the results of a coagulation profile and platelet count were unremarkable. unfortunately, platelet aggregation studies were not done. other causes for coagulation defects include decreased platelet production by infiltrated marrow, increased platelet consumption, or increased platelet destruction. just over half of the cats in this study had thrombocytopenia, 1 of which also had prolonged ptt. increased susceptibility to infection is common in human patients with multiple myeloma, and is potentially lifethreatening. 66 infectious processes in this study included 1 cat each with severe periodontitis, chronic recurrent upper respiratory infections, and terminal bacteremia. multiple myeloma-associated immunodeficiency is likely a multifaceted phenomenon secondary to decreased production of functional ig, 67 suppression of normal b-cell differentiation and function 68 in response to antigenic stimulation, 69 increased rate of gamma globulin catabolism, or neoplastic infiltration of bone marrow resulting in leukopenia. 70 unusual cases in our study included 2 cats with cutaneous plasma cell tumors. solitary cutaneous plasma cell tumors usually are not aggressive, and reports of metastasis are rare. 46, 71, 72 one cat not only had metastasis but met the criteria for both multiple myeloma and plasma cell leukemia (pcl). pcl is an extremely rare disease in any species, and there have been no reports to date in cats, other than this case, published previously as a single case report. 73 two forms of pcl are defined in people: primary, in which there is no previous diagnosis of myeloma, and secondary, in which pcl is a late manifestation of previously diagnosed multiple myeloma or plasmacytoma (as seen in cat 8). regardless of whether primary or secondary, diagnosis of pcl requires !20% marrow plasmacytosis and .2000 plasma cells/ll in peripheral blood. cat 8 met these criteria. in a study of 826 human myeloma patients, plasma cells were observed in peripheral blood smears of 16%, but most of these failed to meet the criteria for pcl. 63 similarly, we had 2 cases with circulating plasma cells, but in insufficient numbers to warrant a diagnosis of pcl. plasma cell leukemia in human patients is associated with a very poor prognosis because it follows a course similar to other acute leukemias 74 and is poorly responsive to chemotherapy. 75 in summary, the most common abnormalities seen in this retrospective study of cats with multiple myeloma included paraproteinemia, bone marrow plasmacytosis, atypical plasma cell morphology, hypocholesterolemia, bone lysis, anemia, and multi-organ involvement. a moderate number of cats had light chain proteinuria, thrombocytopenia, and neutropenia. rare abnormalities include hvs, hypercalcemia, hypoalbuminemia, cutaneous tumors, and pcl. our findings suggest that successful analysis of bone marrow aspirates and serum protein concentrations should be sufficient to establish a diagnosis of multiple myeloma in cats; however, we advocate modifying the diagnostic criteria to include visceral organ infiltration and atypical plasma cell morphology, especially when the percentage of plasma cells in the marrow is ,20%. allowance for atypical morphology or detection of plasmacytosis at additional sites may assist in the diagnosis of otherwise difficult cases. feline multiple myeloma: literature review and four case reports managing the veterinary cancer patient an unusual presentation of multiple myeloma in two cats multiple myeloma in cats: variable presentation with different immunoglobulin isotypes in two cats multiple myeloma in a cat serum hyperviscosity syndrome associated with multiple myeloma in two cats immunoglobulin a myeloma in a cat with pleural effusion and serum hyperviscosity serum hyperviscosity syndrome associated with igg myeloma in a cat multiple myeloma in a cat with paraparesis what is your diagnosis? multiple osteolytic myeloma with fracture plasma cell myeloma producing an immunoglobulin a paraprotein in a cat a case of multiple myeloma in a cat hypercalcemia in two cats with multiple myeloma a case of feline igamonoclonal gammopathy associated with bence jones proteinuria diagnosis and management of monoclonal gammopathies plasma cell myeloma marrow diagnosis including morphologic and phenotypic features plasma cell neoplasms reactive plasmacytic lesions of the bone marrow henry bence jones-physician, chemist, scientist and biographer: a man for all seasons multiple myeloma vertebral plasma cell myeloma causing posterior paralysis in a horse plasma cell myeloma in the horse: a case report and literature review hemorrhagic diathesis caused by multiple myeloma in a three-month-old foal monoclonal gammopathy in a horse with defective hemostasis serum protein changes in four horses with monoclonal gammopathy multiple myeloma in a horse multiple myeloma in a horse characterization of a homogeneous paraprotein from a horse with spontaneous multiple myeloma syndrome monoclonal aggregating immunoglobulin cryoglobulinaemia in a horse with malignant lymphoma review of 1027 patients with newly diagnosed multiple myeloma prognostic factors for multiple myeloma in the dog myeloma aetiology and epidemiology multiple myeloma and human immunodeficiency virus-1 (hiv-1) infection bone marrow immunohistology of plasma cell neoplasms hepatic plasmacytoma and biclonal gammopathy in a cat monoclonal gammopathy in a dog with visceral leishmaniasis monoclonal gammopathies in the dog: a retrospective study of 18 cases (1986-1999) and literature review monoclonal gammopathy associated with naturally occurring canine ehrlichiosis canine monocytic ehrlichiosis presenting as acute blindness 36 months after importation into the uk canine ehrlichiosis in six dogs with persistently increased antibody titers monoclonal gammopathy in a dog with chronic pyoderma a case of feline macroglobulinemia a benign hypergammaglobulinemia mimicking plasma cell myeloma monoclonal gammopathies of undetermined significance a long-term study of prognosis in monoclonal gammopathy of undetermined significance plasma cell tumors in a cat animal models of bone metastasis lytic lesion in the distal humerus of a dog aneurysmal bone cyst in a rib of a cat steroid injection therapy in a feline solitary bone cyst aneurysmal bone cyst in a sixmonth-old dog aneurysmal bone cyst in a dog pathological femoral fracture due to rhabdomyosarcoma in a cat rickets caused by excessive renal phosphate loss and apparent abnormal vitamin d metabolism in a cat characterization of osteopenia in feline mucopolysaccharidosis vi and evaluation of bone marrow transplantation therapy osteopenia and other radiographic signs in canine hyperadrenocorticism nutritional secondary hyperparathyroidism in six cats hypercalcemia in cats: a retrospective study of 71 cases (1991-1997) monoclonal immunoglobulin g cryoglobulinemia and multiple myeloma in a domestic shorthair cat effect of iodinated water soluble contrast media on urinary protein assays the clinical significance of bence jones proteinuria prevalence and clinical characteristics of a high cardiac output state in patients with multiple myeloma multiple myeloma: review of 869 cases complications of multiple myeloma plasma cell neoplasms infections in multiple myeloma the role of suppressor cells in the pathogenesis of common variable hypogammaglobulinemia and the immunodeficiency associated with myeloma humoral immune deficiency in multiple myeloma patients due to compromised b-cell function immune function in patients with multiple myeloma (delayed cutaneous reactivity and lymphocytes bearing receptors for sheep, human and mouse erythrocytes) immunosuppression and infection in multiple myeloma cutaneous plasmacytomas in dogs: a morphologic and immunohistochemical study prognostic value of histopathological grading in canine extramedullary plasmacytomas progression of a solitary, malignant cutaneous plasma-cell tumour to multiple myeloma in a cat plasma cell leukemia: a report on 15 patients primary plasma cell leukaemia: a report of 18 cases the authors gratefully acknowledge drs kara stover, john richter, and adrianna richter for providing case material and dr thomas van winkle for assistance with necropsy record retrieval. since acceptance of this article, 4 additional cases of feline multiple myeloma were diagnosed at mjr-vhup. all 4 had hepatic involvement, and 3 had splenic involvement (the spleen of 1 cat was not evaluated). these observations emphasize the advantage of assessing extramedullary sites in cats suspected of having multiple myeloma. furthermore, all 4 cats were hypocholesterolemic, 2 were hypercalcemic, and 1 had multiple lytic lesions, similar to the cats in this study. key: cord-284208-8fsqgkw5 authors: zolla, lello title: proteomics studies reveal important information on small molecule therapeutics: a case study on plasma proteins date: 2008-11-07 journal: drug discov today doi: 10.1016/j.drudis.2008.09.013 sha: doc_id: 284208 cord_uid: 8fsqgkw5 the most abundant proteins in serum, such as albumin and igg, act as molecular sponges that bind and transport low molecular weight proteins/peptides and drugs. in the near future, pharmacoproteomics, the use of proteomic technologies in the field of drug discovery and development, and interactomics, the branch of proteomics which is concerned with identifying interactions between proteins, will allow researchers to (i) know the specific protein changes that occur in biological compartments in response to drug administration; (ii) design small novel therapeutic molecules that can have extended half-lives if carried by plasma protein in the blood stream. advances in these fields will open new avenues of tailor-made molecular therapy, reducing present limitations on treatment arising from toxicity and inefficiency. in this short review we report and discuss the most recent developments arising from the use of proteomic tools in blood plasma protein research, looking at the identification of proteins found in plasma as well as their interactions with small molecules such as drugs, peptides, organic chemicals and metals. we believe this research demonstrates that proteomic technologies, and in particular pharmacoproteomics, interactomics and post-translational modification analysis, could be instrumental in the design of new tailor-made drugs leading to substantial improvements in molecular therapy. proteomics studies reveal important information on small molecule therapeutics: a case study on plasma proteins lello zolla department of environmental science, university of tuscia, piazzale università, viterbo, italy the most abundant proteins in serum, such as albumin and igg, act as molecular sponges that bind and transport low molecular weight proteins/peptides and drugs. in the near future, pharmacoproteomics, the use of proteomic technologies in the field of drug discovery and development, and interactomics, the branch of proteomics which is concerned with identifying interactions between proteins, will allow researchers to (i) know the specific protein changes that occur in biological compartments in response to drug administration; (ii) design small novel therapeutic molecules that can have extended half-lives if carried by plasma protein in the blood stream. advances in these fields will open new avenues of tailormade molecular therapy, reducing present limitations on treatment arising from toxicity and inefficiency.in this short review we report and discuss the most recent developments arising from the use of proteomic tools in blood plasma protein research, looking at the identification of proteins found in plasma as well as their interactions with small molecules such as drugs, peptides, organic chemicals and metals. we believe this research demonstrates that proteomic technologies, and in particular pharmacoproteomics, interactomics and post-translational modification analysis, could be instrumental in the design of new tailor-made drugs leading to substantial improvements in molecular therapy. blood plasma is the most complex human-derived proteome. because of this complexity, and the enormous range of concentration encountered across the population of protein components, spanning in excess of ten orders of magnitude, whole blood plasma is the most difficult specimen to analyze, and this creates serious challenges for proteomics. much progress has already been made in this field and new directions have been put forward and discussed as part of the hupo plasma proteome project (ppp), to focus efforts on the remaining challenges [1] . the ppp initiative has three stated long-term goals (i) to make a comprehensive analysis of the protein constituents of plasma; (ii) to determine the extent and source of variation in an individual's plasma over time; and (iii) to determine the extent of variation in plasma between individuals within and across populations [2] . blood plasma is known to contain proteins derived from blood cells and other body tissues that may have ended up there through cell death or damage (causing proteins to be released from normal cells), or they may come from aberrant protein secretions from tumor cells. in a recent investigation [3] , the examination of the plasma protein component categories revealed that many of the proteins detected in plasma are normally associated with cells (i.e. they are not known plasma proteins). these 'cellular leakage proteins' were categorized according to their original location and function. intracellular proteins accounted for up to 42% of the proteins identified, while membrane-associated proteins, including those proteins that are membrane-based but not known to be released in plasma (i.e. receptors, coreceptors and adhesion molecules) [3] accounted for another 13%. another 5% of the proteins were found to be of cellular origin, and are either secreted or occupy an extracellular location, and 3% were identified as specific cytokines or cytokine-related proteins. all these proteins are generally considered passenger proteins (some more transient than others) that utilize plasma for transportation, localization and mediation of cellular responses. overall, this group is the least characterized but possibly the most interesting one in terms of potential to yield biomarkers, with protein concentrations believed to range from low mg/ml to pg/ml levels, and possibly extending to levels below the detection limits of traditional elisa assays (1 pg/ ml). the 'classic' plasma proteins, those whose activity is specifically localized in the plasma, such as human serum albumin (hsa), complement components and apolipoproteins, make up only 4% of the total protein found in plasma. approximately 34% of the proteins identified, however, had no known function [3] . hence, with such a diverse population of proteins derived from a range of sources we might expect that the analysis of the extracellular proteome of proteins circulating in the plasma and the cell-based proteome are necessary and complementary for an exhaustive plasma investigation. one strategy applied in several recent examples involves using a secondary tissue or fluid of interest to first identify potential candidates for biomarkers and then screening the complementary plasma sample for their presence. such an investigation is highly desirable because disease markers present in plasma may include proteins with significant potential for early disease diagnosis, containing information that directly reflects pathophysiological states and represents an invaluable source of diagnostic information for a variety of different diseases [4] . thus, a broad inventory of plasma proteins (both qualitative and quantitative) could be used for the identification of putative protein markers for any diagnosable disease as well as for the development of new therapeutic products [4] . quantitatively speaking, the core plasma protein is albumin, representing about 50% of the total plasma protein content (in the order of 30-50 g/l). immunoglobulins (igs) represent 20-25% of the total protein mass [3] . low-abundance plasma proteins from tissue leakage and cytokines are present in the range of picograms to nanograms per milliliter. the most abundant proteins in serum, such as albumin, igg and transferrin, are known to act as the carriers for hormones, lipoproteins and many other proteins, lipids and metals. they may, in fact, be described as molecular sponges, which bind and transport low molecular weight (lmw) protein/peptide species preventing their rapid clearance by the renal system, thereby extending their half-life in the blood stream. although it has been estimated that 32% human plasma proteins do not display associations with the major proteins, about 48% have been found in association with igg and 20% of proteins have also been found with hsa codepleted groups, suggesting the possibility of weak overlapping interactions between some proteins and both hsa and igg (see fig. 1 ). moreover, because proteins in the circulatory system are exposed to a variety of proteases or chemical oxidations [5, 6] , plasma is rich in peptides, the so-called 'peptidome' (see box 1) from which about 5000 peptides have been revealed [7] , prevalently associated with the carrier proteins. if igs are retained in the sample during peptide-based proteomic studies, they can act as a generous source of random peptide sequences contributing to a large library of 'background' species that, because of their abundance (20 mg/ml sample concentration) and variety, greatly complicate the process of peptide detection and identification. plasma protein interactions appear to exhibit little dependence on protein size. in fact, small plasma proteins such as the basic proline-rich peptide p-e (ib-9, mw = 6020 da, igg codepleted protein) show more interaction specificity than large proteins such as cardiac titin isoforms (mw = 2,991,181 da) that are typically found in more than one protein interaction group [3] . this kind of information, concerning the interactions between human plasma proteins, should be useful for further studies in human blood systems/network biology, such as evaluating possible protein contaminants in therapeutic protein products prepared from human plasma, and for the design of analytical approaches to deplete high abundance plasma proteins effectively. additionally, it could be used to help design proteins with a reduced rate of clearance from the circulatory system, particularly important when small proteins are used as therapeutic agents [8] . normally, small uncomplexed proteins and peptides (i.e. less than 40 kda) are rapidly cleared from the circulation through enzymatic degradation, uptake by the reticuloendothelial system or by glomerular filtration, which discriminates on the basis of molecular size and charge [8] . it is believed that the circulation half-life of this lmw fraction is directly related to its binding affinity to large high abundant carrier proteins. drug discovery today volume 13, numbers 23/24 december 2008 box 1 the array of peptides normally present within the circulatory proteome is termed the 'peptidome' , and could be a rich source of cancer-specific diagnostic information because it provides a record of the cellular and extracellular enzymatic events that take place at the level of the cancer-tissue microenvironment. this new information archive seems to show that most peptides in vivo are bound to high-abundance proteins such as albumin. measuring panels of peptidome markers might be more sensitive and specific than conventional biomarker approaches. a biomarker is a biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or a disease. a biomarker may also be used to see how well the body responds to a treatment for a disease or condition, to measure the progress of disease or the effects of treatment. the use of proteomic technologies in the field of drug discovery and development. toxicoproteomics is the use of global protein expression technologies to better understand environmental and genetic factors, both in the episodes of acute exposure to toxicants and in the long-term development of disease. primary aims in toxicoproteomics are the discovery of key-modified proteins, the determination of affected pathways, and the development of biomarkers for eventual prediction of toxicity. the use of proteomic technologies and tools, such as phosphorylation-site-specific antibodies, to assess and monitor the phosphorylation state(s) of various proteins involved in signaling pathways in cells. a method that allows for the quantitative measurement of proteins in two separate populations by mass spectrometry via the differential tagging of proteins in each population at cysteine residues with heavy or light isotope reagents. protein microarrays are composed of a series of immobilized spots, containing a homogeneous or heterogeneous 'bait' molecule. a spot on the array can represent an antibody, a cell or phage lysate, a recombinant protein or peptide or a nucleic acid. the array is queried with either a probe (a labeled antibody or ligand), or an unknown biological sample (e.g. a cell lysate or serum sample) containing analytes of interest. by directly or indirectly tagging the query molecules with a signal-generating moiety, a pattern of positive and negative spots is generated. a combinatorial ligand library is composed of millions of diverse hexapeptide baits, able to capture aspecifically all peptides/ proteins in any given proteome. in this way they concentrate the 'low-abundance' proteome, while drastically cutting the concentration of the most abundant compounds. it is based on the concept of a 'one-bead, one-peptide' approach. free-flow electrophoresis (ffe) is electrophoresis carried out in an aqueous medium without using any solid matrix, such as acrylamide. it is useful for the separation of a wide variety of charged analytes like low-molecular weight organic compounds, peptides, proteins, protein complexes, membranes, organelles and whole cells in aqueous media under native and denaturing conditions. the analyte is injected into a thin, laminar separation buffer film (which defines the electrophoretic mode such as zone electrophoresis, ief or isotachophoresis) and is deflected by an electric field perpendicular to the flow direction. differential scanning calorimetry differential scanning calorimetry measures the difference in the amount of heat required to increase the temperature of a changed biological sample with respect to an unchanged one (reference) as a function of temperature. the difference in thermograms of blood plasma between normal and diseased individuals is not related to the concentrations of the most abundant plasma proteins but, rather, seems to arise from binding interactions involving as yet unknown biomarkers with the more abundant plasma proteins, particularly albumin. such a behavior is consistent with the 'interactome' hypothesis. interactomics is a fusion science of biology, informatics and engineering which provides a global view of protein family interaction networks. it involves the study of both the interactions and the consequences of those interactions between and among proteins, and other molecules within a cell and can be used to compare networks of interaction between and within species to see how the traits of such networks are varied and conserved. the interactome network includes certain calculated parameters that weigh the reliability of a given interaction (i.e. the 'edges' of the interactome network) between two proteins, and also qualify the functional environment around any given protein (i.e. the 'nodes' of the interactome network). some of the strategies devised to retard the clearance of therapeutic proteins include the covalent attachment of polyethylene glycol or dextran chains or by protein-protein crosslinking. genetic modification has also been used to create chimeras of the therapeutic protein of interest with long-lived plasma proteins like albumin or igg [9] . over 10,000 different proteins have been estimated to be commonly present in the plasma, most of which are at very low relative abundances [10] . the dynamic range of currently available proteomic techniques such as mass spectrometry (ms) or 2d gel electrophoresis (2-de) spans three to four orders of magnitude and as such does not approach the ten orders of magnitude represented by the plasma proteins. thus, a significant challenge for proteomic analysis of plasma is how to reveal the low-abundance proteins. the strategies that have been most frequently used to overcome the dynamic range problem are to fractionate the plasma proteome into smaller subsets, and/or to deplete one or more of the major proteins. immunoaffinity is the most efficient way to deplete proteins and so these methods are most widely used [11] [12] [13] . immobilized antibodies packed in columns or cartridges capture several the most abundant proteins lowering the protein content down to 1% of the initial amount. although nontarget proteins are removed in this process, it has been shown to be reproducible [14] . multiple orthogonal separation steps have also been used, such as strong cation exchange chromatography followed by reverse phase-liquid chromatography (rp-lc) [8] or insolution isoelectric focusing (ief) [15] . recently, a novel separation approach has been proposed, using 2d free-flow electrophoresis (ffe), separating proteins and peptides in solution according to their pi before lc-ms/ms [16] . a commercial combinatorial ligand library (proteominer) is now available, enabling researchers to pick out the low-abundance proteins [17] . another innovation is the microflow mf10, a device to prefractionate complex low volume, low-abundance samples that can also enrich for very specific species of proteins based on charge and/or size either in native or in denaturing format [18] . all these strategies concomitantly remove proteins/peptides associated with the highly abundant proteins targeted for depletion. a recent study [19] focused on the binding properties of six of the most abundant serum proteins -to investigate the small peptides/proteins that may be bound to them. each of the proteins targeted was found to be capable of binding several different peptide/proteins (210 proteins from a total of 378 unique peptides), many of which are clinically useful biomarkers. these results suggest that, on the one hand, albumin or igg depletion before protein identification may actually eliminate many valuable biomarkers but, on the other hand, the identification of biomarkers, through the selective isolation of protein bound to the more abundant proteins in serum, could be a novel proteomic strategy. because the general and specific sample losses increase with each separation stage, the best solution to this dilemma is to maximize the efficiency of each separation stage and minimize several dimensions required for the characterization of complex mixtures. as stated above, proteomic approaches may be utilized for disease classification as well as for the development of novel biomarkers related to prognosis, diagnosis and choice of therapeutic regimen. it is generally accepted that these biomarkers will not originate from classic plasma secretions but, most probably, from leakage, secretion or shedding of proteins from the specific affected tissue, cell type or cellular pathway. nevertheless, blood is still the logical choice of biospecimen and has become the most frequently used biomarker discovery matrix to date, because the driving force of biomarker discovery is the development of blood-based assays for early detection and prediction of therapeutic response [20] . it follows that if protein biomarkers have been identified in biopsied tissues, researchers will then be able to examine the blood to determine whether these biomarkers are in circulation. the great advantage being that blood tests are much less invasive than biopsies. the downside is that once such proteins are released into around 6 liters of plasma, their concentration is extremely low. hence, the need to extend the dynamic range of protein detection and this is where proteomics comes into its own. although blood is a very convenient and noninvasive fluid to monitor for biomarkers, it poses many challenges from the perspective of protein detection. probably the greatest potential application for proteomics lies in investigating pathways that are easily targeted by small molecules or therapeutic antibodies. among the challenges facing clinical proteomics is the ability to link protein expression profiles to specific disease phenotypes and the identification of relevant biomarkers to develop diagnostic tools [21] . in this sense, proteomics is an expansion of reductionist biology, where single proteins are analyzed in a high throughput fashion to arrive at an understanding of the entire system. the intention is that, once the human blood proteome has been fully described, several biomarkers will emerge for each particular disease state that can be used to provide a 'fingerprint' of that disease and here, there are obvious implications for blood donor testing [22] . there are already systematic searches underway to look for plasma proteins that are biological indicators or biomarkers for cancer (see refs. [23, 24] ). if a suite of biomarkers were to be available for early detection, stratification into distinct subtypes and monitoring of progression or response to therapy we could expect significant improvements in clinical outcomes for cancer patients. the intention is to develop panels of biomarkers that will allow early detection of cancer and prediction of the probable response to therapy, possibly detectable in a single proteomics experiment. despite the recent progress in proteomic technologies based on ms, however, the discovery of novel clinical assessment tools has been slow. this is partly because of the inherent difficulties in working with blood. it is hoped that a better understanding of the limitations of blood for comparative protein profiling and an appreciation of the advantages of cancer tissue or cancer cell secretomes will greatly enhance the progress [25] . a comparative proteome study of the body fluids of schizophrenia patients has been carried out to look for biomarkers or associated proteins in an effort to understand the etiology of schizophrenia. it was found that protein expression of the ttr tetramer and apolipoprotein e (apoe) was downregulated by up to 1.68 and 3.62 times, respectively, in schizophrenia patients compared to normal controls [26] . a study that revealed potential diagnostic cancer biomarkers used blood from sjl (selected by james lambert) mice, in which the growth of rcsx lymphoma cells induces an inflammatory response by stimulating v b 16+t cells [27] . after the depletion of albumin and immunoglobulin, 1079 mouse nr proteins were identified in sjl mouse plasma in a single experiment. most were found to be upregulated (e.g. acute phase reactants) but some proteins were found to be unique to the tumor-bearing mouse plasma (i.e. haptoglobin, proteosome subunits, fetuin-b, 14-3-3 zeta and mage-b4 antigen). while it remains to be seen whether a similar unique profile occurs in human lymphomas, recent studies have demonstrated that in sepsis and cancer, the interalpha inhibitor proteins (iaips), a family of structurally related serine protease inhibitors, are present in relatively high concentrations in human plasma, suggesting their suitability as potential biomarkers [28] . it would seem logical to assume that a better understanding of the biological mechanisms of drug toxicity and the development of therapeutic resistance would lead to the development of improved therapeutics with significant utility in clinical research. in this context, pharmacoproteomics, the use of proteomic technologies in the field of drug discovery and development (see fig. 2 ), is an emerging science. protein drug-activity biomarkers can be defined as specific protein changes in total protein content in biological compartments that occur in response to drug administration [29] . in contrast to genomic approaches, the application of pharmacoproteomics, by virtue of being stimulus-induced, may be more amenable to systematic experimental manipulation that could ultimately result not only in validation of a biomarker, but also in characterizing its behavior under specific conditions [30] . in association with chemical compound library screening strategies, pharmacoproteomics is expected to accelerate the discovery of new lead compounds for future drugs. it should also play an important part in preclinical studies by providing information on the mode of action and the eventual deleterious side effects of drugs. hopefully, ongoing and future proteomic studies will open up new avenues of tailor-made molecular therapy, reducing present limitations associated with treatment toxicity and efficiency. ms-based proteomics is ideal in this respect, because it offers a targeted way to identify hundreds of protein or peptide biomarkers simultaneously, with the obvious potential for discovering drugactivity markers because there is often no a priori knowledge of the particular proteins that are likely to change [31] in response to drug administration [32, 33] . while sample enrichment narrows the dynamic range over which molecules of interest must be detected, the improvements in separation technology and ms have extended the ability to find and identify proteins over an ever wider range of relative protein concentrations. ion trap instrumentation has been superseded by the greatly improved linear ion trap mass spectrometers. at the same time, there has been a steady decrease in the diameters of lc-capillary columns that has resulted in increased sensitivity and ability to quantify proteins. this has improved analysis by increasing, by orders of magnitude, the detectable dynamic range of a protein that would not have been picked up previously in samples of similar size. as such, at this time, lc-fticr-ms (fourier transform ion cyclotron drug discovery today volume 13, numbers 23/24 december 2008 theoretical example of differential screening of plasma proteins from healthy, ill and treated (responding and resistant) patients by 2d gel electrophoresis. the appearance or disappearance of protein spots as well as quantitative variations of protein expression can be observed. resonance ms) technology provides the most sensitive and powerful measurement platform and though some significant potential exists for further increasing its dynamic range, this applies to those cases limited by sample complexity, rather than detection limits. recently, however, differential scanning calorimetry has provided a new window into the plasma proteome. thermograms of plasma from diseased individuals not only were found to differ dramatically from those of normal (control)_individuals, but also differed between sufferers of different illnesses, at least for the three diseases examined (arthritis, lyme disease and lupus). each disease appears to have a distinctive and characteristic thermogram. these radically different thermograms seen for different diseased states seem to arise from binding interactions involving as yet unknown biomarkers with the more abundant plasma proteins, particularly albumin. such behavior is consistent with the 'interactome' hypothesis, suggesting a novel use for calorimetry as a diagnostic tool [34] . for a careful approach to clinical design and data analysis it is important to maximize the chance of discovering meaningful drug-activity markers. the first step is to establish a link between drug administration and the resulting biologically quantified change [35] . this strong foundation should set the stage for any data-driven hypothesis generation and testing, and further establish the utility of such a multidisciplinary tool in expanding the frontiers of clinical research. too often there is no information available on the tissue under investigation, in these circumstances protein-profile changes in response to drug treatment can serve as evidence of cell pharmacodynamic activity, and such data can be used to establish a minimum dose for early clinical studies. more broadly, because targeted proteomics can also be hypothesis generating, detecting a drug-induced change can also lead to new lines of research and inquiry in the field of drug development or pathophysiology. a clear understanding of proteomic variability under controlled conditions is crucial to enable future clinical studies to be appropriately designed and to improve the contextual interpretation of data obtained from pharmacoproteomic studies. it is also important to establish a suitable time period between drug administration and analysis primarily to allow sufficient time for a tissue-targeted drug to reach a pharmacokinetic steady state before exploring protein profile changes [36] . this is to ensure minimal interference from extraneous factors on endogenous variability, thus maximizing the probability of identifying even subtle drug-induced changes. the variability between individual subjects could potentially affect observations in unpaired comparison studies, such as diseased versus healthy volunteers, and should be taken into account [37] . in other words, we cannot assume that pharmacodynamic responses to identical chemical or biochemical stimuli will be similar among healthy individuals and that these responses will differ significantly and consistently in patients with pathological disorders, because a variety of factors will influence the profile of each individual [37] . this aspect requires further investigation to ascertain how useful biomarkers will ultimately prove to be and what consequence this will have for drug development. pharmacoproteomics is not limited to the extracellular proteome analysis of blood plasma, because cell-based proteome analysis also has an important role in this field. in this context, post-translational modifications (ptm) of proteins have a significant impact. some 300 potential modifications are known, and they include phosphorylation, glycosylation, acetylation, myristoylation, palmitoylation, methylation, sulfation, prenylation and ubiquitylation (http://www.abrf.org/index.cfm/ dm.home). during the past two decades, sensitive ms methods have been refined to determine the type and site of protein modifications that can seldom be predicted from a genomic sequence [38] . some of the protein modifications are regulatory and reversible, most notably phosphorylation which controls protein functions such as localization, complex formation, stability and activity through different mechanisms [39] . as a result of extensive research, we now know that about 30% of all human proteins can be modified by phosphorylation, while only 0.05% of the phosphorylated residues are tyrosine [40] . protein phosphorylation is one hallmark of the protein-protein interactions (ppis) underlying signaling networks and, in many cases, it is aberrant protein kinase activity that drives diseaseassociated derangements in signaling pathways. the aim would be to design drugs that effectively disrupt this aberrant proteinphosphorylation-based enzymatic activity and epigenetic phenomena. pharmacoproteomics, or the tailoring of therapy based on proteomic knowledge, is expected to take a central role in this process [41] . a recent study describes a cell-based drug discovery platform based on phosphospecific flow cytometry, or phosphoflow, with which researchers were able to screen for inhibitors of multiple endogenous kinase signaling pathways in heterogeneous primary cell populations at the single-cell level [42] . protein microarrays that examine protein-protein recognition events (i.e. phosphorylation) in a global, high-throughput manner have been used to profile cellular signal pathways in a way not possible with gene arrays [43, 44] . the activity levels of the proteins in a particular pathway can thereby be assessed in 'real time', to tailor treatment to each patient's cellular 'circuitry' [45] [46] [47] . the advantage of protein microarrays lies in their ability to provide a 'map' of known cellular signaling proteins that generally reflect the state of information flow through protein networks in individual specimens. the identification of crucial nodes or interactions within the network (see later) is a potential starting point for drug development and the design of individual therapeutic regimens [48] [49] [50] . accurate annotation of peptide modifications through unrestrictive database searches can reveal post-translational modifications, as well as sequence polymorphisms [51] . monitoring different phosphoprotein levels will also help to identify treatment-acquired resistance to chemotherapy. the effect of imatinib on the tyrosine phosphoproteome in bcrabl positive leukemia cell lines has been examined by proteomic approaches. the investigation revealed 64 sites of tyrosine phosphorylation corresponding to 32 different proteins [52] . affinity column chromatography using an immobilized pyrido(2,3-d)pyrimidine derivative has been successfully used to select protein kinases that bound to the matrix and were then identified using ms, demonstrating the fact that pyrido(2,3-d)pyrimidine is a kinase inhibitor with low specificity [53] . a similar methodological approach was applied to study the p38 kinase inhibitor sb203580, demonstrating that cyclin g-associated kinase (gak) and ck1 were almost as potently inhibited as p38a [53, 54] . protein expression subsequent to treatment with the hdaci trichostatin-a has been studied in human pancreas ductal carcinoma cell lines using 2-de and maldi-tof ms [55, 56] . trichostatin-a appears to upregulate proteins which promote cell death and downregulate proteins that favor cell growth. similarly rc307 modulates proteins that are involved in proliferation, cell cycle regulation, apoptosis and gene expression in colon carcinoma cells [57] . the pharmacoproteomic approach was found to be particu-larly useful for the identification of molecular alterations implicated in type 2 diabetes, and for further characterization of existing or new drugs [58] . research has shown that several physiological factors lead to changes in both the plasma proteome and the peptidome, including stress, sleep, sport training, eating meals and pregnancy. exposure to toxic agents, including drugs, can also be detected using proteomics leading to a distinct field of study known as toxicoproteomics [59] . recently, the proteins in the plasma of workers exposed to benzene were analyzed [60] . two drug discovery today volume 13, numbers 23/24 december 2008 human erythrocyte protein-protein interaction network (interactome). nodes of the network are the known rbc proteins and links connecting the nodes correspond to the known interactions [70] . significantly upregulated protein profiles were found in workers exposed to the toxic chemical: the t cell receptor b chain and matrix metalloproteinase-13. thus, the plasmatic t cell receptor b chain may be a useful indicator for the early detection of exposure to benzene. comparative proteome analysis of human plasma allowed qian et al. [61] to identify 32 proteins that were significantly increased after lipopolysaccharide administration. a study of the plasma of workers occupationally exposed to polycyclic hydrocarbons revealed several proteins as markers of such an exposure [62] . using the same technique, truncated forms of a1-antitrypsin were discovered in serum samples of patients presenting with severe acute respiratory syndrome [63] . in another study involving patients with the same syndrome, samples revealed a total of 38 differential spots, most of them corresponding to acute phase proteins [64] . the authors also identified proteins such as peroxiredoxin ii, not previously detected in plasma 2-de. the investigation of plasma protein interactions with metals remains a relatively new and challenging arena for toxicoproteomics. current studies include the identification of the protein carrier and how to determine toxic metal concentrations, which requires sensitive analytical techniques and powerful equipment. thankfully, the search for suitable quantitative techniques in the field of drug design and proteomics may be almost over with the development of inductively coupled plasma ms (icp-ms) [65] . another relevant field for pharmacoproteomic applications is that of elucidating the biological mechanisms of drug resistance. in drug-resistant cells alternative protein forms appear that prevent the drug binding to active sites and/or executing signaling effects independent of the regulated native protein forms. the methodology of proteomics seems highly applicable to the search for drug-resistant protein forms (drug-resistance proteomics). in this context the plasma proteome in aspirin (acetylsalicylic acid [asa])-sensitive and asa-resistant coronary ischemic patients has been analyzed. the expression of one isotype of the fibrinogen g chain and three isotypes of haptoglobin was increased in asa-resistant patients as well as that of three vitamin d binding protein (dbp) isotypes. it has been suggested that dbp regulates the inhibitory effect of asa on platelets, by reducing the inhibitory effect of asa on thromboxane a2 production [66] . in the field of oncology, proteomics is becoming widely used for the identification of tumor-specific protein markers, and pharmacoproteomics has found a place in the evaluation of chemotherapy, particularly for the characterization of drugresistance mechanisms [58] . identifying important interactions between blood proteins is emerging as another focus of blood-based proteomic research. many proteins circulate in blood, not as single entities but as multicomponent complexes, and as such comprise the blood 'interactome' [4] . protein-protein interactions (ppi) information can be extracted from the currently available databases of interactions (in silico approaches). little is currently known about the interactions of any given protein in the blood or whether these interactions are biologically relevant. examining protein complexes will, however, often yield information about protein function. the identification of such interactions may bring to light important information for designing new small molecule therapeutics. this conceptual framework contrasts with the single protein (or single pathway) approach that has previously dominated biology. the combination of proteomic and in silico approaches allows one to not only identify disease and/or drug-related changes in the proteome but also predict the changes in the protein interactome network that are associated with disease-related change of function. it can also predict modifications associated with ptm changes occurring as a result of a specific disease or stage of disease, as well as drug or gene therapy treatment. these linked approaches represent the future of clinical identification of a specific disease, its current stage or severity, and the effects and side effects of various treatments. clearly, an interactome of plasma proteins is far from reality at present, because of its complexity, while the simplicity of the human erythrocyte cell structure has instead made it an optimal cell for proteomic study. in fact, while nucleated cells contain 20,000-30,000 proteins [31, 67, 68] , red blood cells (rbcs), which lack nuclei and other organelles, contain far fewer. a comprehensive list of rbc proteins known to date contains 751 entries obtained using proteomics technology [69] . the resulting ppi network is depicted in fig. 3 [70] . the network presented here was derived from protein interaction data obtained from the unified human interactome (unihi) [71] . the correlation reported by unihi is derived from gene expression experiments and represents a measure of confidence for the interaction [72] . as the knowledge of ppis continues to increase owing to advances in proteomics research we expect that, in addition to a deeper understanding of the erythrocyte protein complement and how it relates to function, it will also be possible to understand how changes in the rbc proteome and interactome affect the development of erythrocyte disorders [69] . the promise of proteomic-based profiling, as opposed to gene transcript profiling alone, is that the resulting prognostic signatures are derived from drug targets (proteins) not genes, so the pathway analysis provides a direction for therapeutic mitigation. the only dark cloud on the horizon may be that extending the use of pharmacoproteomics (beyond molecular network analysis for patient-tailored therapy to include risk stratification or 'predispostional testing') is likely to raise a larger societal issue, particularly in a climate of cost containment. the powerful combination of pharmacoproteomics and interactomics may win through despite costs, furnishing new targeted therapies for disease prevention as well as for posttherapy monitoring. in short, these techniques not only provide a window on the presence or severity of a specific disease, and drug and gene therapy treatment, but can also be used to identify disease-and/ or drug-related changes in the proteome. they can also predict those changes in the protein interactome network that are associated with disease-related changes of function [73] . without doubt, these linked approaches represent the future for the cost effective and noninvasive identification of specific clinical diseases, their prognosis and effective treatment. hupo plasma proteome project: challenges and future directions the human proteome organization plasma proteome project pilot phase: reference specimens, technology platform comparisons, and standardized data submissions and analyses characterization of the human blood plasma proteome utilizing human blood plasma for proteomic biomarker discovery oxidation of proteins: basic principles and perspectives for blood proteomics proteomic analysis of rbc membrane protein degradation during blood storage composition of the peptide fraction in human blood plasma: database of circulating human peptides peptide and protein drug delivery to and into tumors: challenges and solutions effects of genetic engineering on the pharmacokinetics of antibodies toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry multi-component immunoaffinity subtraction chromatography: an innovative step towards a comprehensive survey of the human plasma proteome differences among techniques for high-abundant protein depletion immunoaffinity separation of plasma proteins by igy microbeads: meeting the needs of proteomic sample preparation and analysis evaluation of multiprotein immunoaffinity subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry a novel four-dimensional strategy combining protein and peptide separation methods enables detection of low-abundance proteins in human plasma and serum proteomes two-dimensional separation of human plasma proteins using iterative free-flow 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myeloid leukemia (aml) pharmacogenomics and pharmacoproteomics in the evaluation and management of short stature from genomics to proteomics mass spectrometry-based proteomics mass spectrometry-based clinical proteomics calorimetry outside the box: a new window into the plasma proteome the impact of systems approaches on biological problems in drug discovery identifying pharmacodynamic protein markers of centrally active drugs in humans: a pilot study in a novel clinical model alpha cell function in health and disease: influence of glucagon-like peptide-1 technologies and methods for sample pretreatment in efficient proteome and peptidome analysis quantitative phosphoproteomic analysis of signaling network dynamics signaling -2000 and beyond technology insight: pharmacoproteomics for cancerpromises of patient-tailored medicine using protein microarrays high-content single-cell drug screening with phosphospecific flow cytometry antibody arrays in cancer research protein chip technology reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front progress in protein and antibody microarray technology protein microarrays as tools for functional proteomics experimental design and analysis of antibody microarrays: applying methods from cdna arrays clinical proteomics: translating bench side promise into bedside reality protein biochips: a new and versatile platform technology for molecular medicine accurate annotation of peptide modifications through unrestrictive database search profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry chemical proteomic analysis reveals alternative modes of action for pyrido2 3-dpyrimidine kinase inhibitors an efficient proteomics method to identify the cellular targets of protein kinase inhibitors proteomic analysis of pancreatic ductal carcinoma cells treated with 5-aza-2 0 -deoxycytidine proteomic analysis of pancreatic endocrine tumor cell lines treated with the histone deacetylase inhibitor trichostatin a a proteomic approach for evaluating the cell response to a novel histone deacetylase inhibitor in colon cancer cells pharmacoproteomic approach to the study of drug mode of action, toxicity, and resistance: applications in diabetes and cancer toxicoproteomics: serum proteomic pattern diagnostics for early detection of drug induced cardiac toxicities and cardioprotection proteomic analysis of plasma proteins of workers exposed to benzene comparative proteome analyses of human plasma following in vivo lipopolysaccharide administration using multidimensional separations coupled with tandem mass spectrometry protein biomarkers in the plasma of workers occupationally exposed to polycyclic aromatic hydrocarbons the use of proteomics in the discovery of serum biomarkers from patients with severe acute respiratory syndrome plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry the application of inductively coupled plasma mass spectrometry in pharmaceutical and biomedical analysis relationship between vitamin d binding protein and aspirin resistance in coronary ischemic patients: a proteomic study the abc's (and xyz's) of peptide sequencing proteomics of organelles and large cellular structures the human red blood cell proteome and interactome centrality measures for the human red blood cell interactome unihi: an entry gate to the human protein interactome a graph theoretic approach to testing associations between disparate sources of functional genomics data transfusion medicine in the era of proteomics this work was supported in part by the italian national blood centre, istituto superiore di sanità, rome, italy (directed by dott. giuliano grazzini). the author would like to thank dr g.m. d'amici for his help and dr j. scarpa for english revision. key: cord-018492-d34tyar7 authors: zaza, mouayyad; kalkwarf, kyle j.; holcomb, john b. title: dried plasma date: 2019-05-06 journal: damage control resuscitation doi: 10.1007/978-3-030-20820-2_8 sha: doc_id: 18492 cord_uid: d34tyar7 dried plasma provides an alternative for early plasma transfusion in the resuscitation of hemorrhagic shock in environments where fresh frozen plasma is not immediately available. it is produced by freeze-drying or spray-drying liquid or thawed plasma. it is shelf-stable for prolonged periods, can be stored at room temperature, and is easy to transport, reconstitute, and administer. it was widely used in wwii but fell out of favor due to the risk of infectious disease transmission. the german and french experiences with lyophilized plasma are the most extensive and show a good track record of efficacy and safety. recent studies show many beneficial effects of dried plasma in the treatment of shock in large animal models. currently, no fda-licensed product is available in the usa, but several are under development. hemorrhage remains the leading cause of preventable death in trauma patients [1] . contrary to the classical teachings of the "golden hour," in patients with severe truncal hemorrhage, peak mortality occurs at 30 min [2] . for patients who survive long enough to make it to a hospital, the median time to death from hemorrhage is 90-150 min after admission [1, 3] . the lethal triad of hypothermia, acidosis, and acute coagulopathy of trauma is well recognized as a common pathway to irreversible shock and death [4, 5] . rapid treatment utilizing damage control resuscitation (dcr) with blood components mimicking whole blood in the setting of permissive hypotension, avoiding crystalloid and colloid, followed by prompt surgical hemorrhage control, is the best strategy to prevent traumatic hemorrhagic death and the onset of the lethal triad [1, 4, 6, 7] . plasma transfusion is an essential part of this approach and remains a significant logistical challenge even today, hence the need for a reliable, shelf-stable, easy-to-carry and administer plasma product such as dried plasma. the role of plasma in dcr has been reaffirmed time and time again. in 1945, beecher noted that "plasma gives more time to get whole blood into the patient" [8] . multiple us large center retrospective cohorts showed that 24-28% of severely injured trauma patients are found to be coagulopathic on admission. coagulopathic patients have significantly higher mortality rates, while the degree of coagulopathy correlated with the severity of injury [9] [10] [11] . analysis of the german trauma registry yielded similar results with rates of coagulopathy up to 34% as well as a direct correlation between the amount of crystalloid received prehospital and an increasing degree of coagulopathy [12] . looking back at the first decade of war in iraq, 33% of combat casualties were coagulopathic on admission, which correlated with a fivefold increase in mortality [13] . these data highlight the importance of recognizing the early onset of the coagulopathy of trauma at the time of injury, as well as endothelial damage, which plasma has been shown to correct [14] . therefore, correction of coagulopathy should not be delayed until laboratory values are available to guide therapy and goals of care. the approach to the trauma patient should aim at preventing and correcting this coagulopathy as soon as possible to decrease its effects on mortality and progression of shock. the importance of balanced resuscitation to include a high ratio of plasma delivered early became evident in the iraq war where a survival advantage was noted in patients receiving close to 1:1 plasma-to-rbc ratio during massive transfusion compared to those who received less plasma [15] . during this conflict, over 335,000 units of blood products were transfused with nearly 110,000 being plasma [16, 17] . analysis of combat casualties over 10 years revealed improved survival in those receiving a higher ratio of plasma and platelets to blood [13] . by 2010, clinical practice guidelines implemented by the department of defense (dod) led to almost 100% of combat massive transfusions to be at a 1:1:1 ratio [18] . the prommtt trial then showed that early plasma administration was associated with reduced mortality in the first 6 h [3] . following that, the proppr trial demonstrated that patients transfused with a higher plasma ratio achieved more hemostasis and had less early death due to exsanguination [19] . a retrospective review of patients receiving thawed plasma available in the ed showed shorter time to plasma transfusion (43 vs. 89 min), a reduction in blood transfused over 24 h, and decreased 30-day mortality [20] . a recent review of us combat casualties in afghanistan rescued by medical evacuation (medevac) units showed that early blood product transfusion prehospital or within minutes of injury resulted in greater 24-h and 30-day survival [21] . in a large civilian cohort from a level 1 trauma center, prehospital administration of blood products including rbcs and plasma has been shown to be feasible and beneficial with improved acid-base status on admission and decreased overall blood product use in 24 h as well as a reduction in the risk of death in the sickest patients over the first 6 h [22] . all these findings point toward the need for a method to deliver early plasma in a reliable fashion. plasma produces superior volume expansion when compared to crystalloids, allowing less volumes infused to match volume lost, faster hemodynamic recovery, and decreased third-spaced volume [23] . it is important to use plasma as the primary resuscitation fluid for patients who are bleeding [4] . other benefits of plasma stem from its ability to mitigate the effects of shock on physiology. the role of plasma in the correction of the endotheliopathy of trauma has been reported in multiple studies and is likely more important than correcting coagulopathy. the mechanism of action is thought to be through promoting systemic vascular stability and preventing endothelial permeability, coagulopathy, and inflammation which eventually lead to shock and end-organ failure [24, 25] . moreover, in a rat model of injury and shock, plasma was able to restore the endothelial glycocalyx, improve syndecan-1 expression, and correct lung injury caused by shock [26] . in a swine model of traumatic brain injury (tbi) and hemorrhagic shock, transfusion of fresh frozen plasma (ffp) decreased neurologic impairment and hastened recovery to baseline cognitive function when compared to saline infusion [27] . neuroprotective effects of plasma in hemorrhagic shock and tbi are thought to be due to improved cerebral perfusion, decreased glutamate-mediated excitotoxicity, and reduction in mitochondrial dysfunction as demonstrated in animal models [28] . plasma also reduced the size of brain lesions and swelling in multiple swine models of tbi and hemorrhagic shock [29, 30] . it also incurs neuroprotection by providing higher brain oxygenation and cerebral perfusion profiles [30] . in humans, a large retrospective cohort analysis showed that in the subgroup of patients with multifocal intracranial hemorrhage, early administration of plasma was associated with a survival benefit [31] . unfortunately, delivery of early balanced resuscitation is fraught with strategic and logistical challenges. in the usa, half of all trauma patients are cared for outside of levels 1 and 2 trauma centers, where blood products are frequently not readily available [32] . in the military, forward surgical teams (fst) were developed to provide immediate support and treatment to injured soldiers. they used to only carry red blood cells (rbcs) for transfusion, and more recently, they are able to provide plasma but not platelets [33] . warm fresh whole blood (wfwb) is available in these circumstances and has been shown to improve survival in combat casualties treated by fst [33] . these challenges, along with misconceptions regarding the role of blood products in resuscitation, led to the overuse of crystalloids and its own set of unique complications. excessive crystalloid resuscitation has been shown to be detrimental, while balanced blood product resuscitation decreases the onset of acute respiratory distress syndrome (ards) and abdominal compartment syndrome and improves survival [32, 34] . the recognition of the importance of early, balanced dcr has led many trauma systems in the usa, and around the world, to search for novel approaches to provide blood products as soon as possible to severely injured trauma patients. these include carrying them in the prehospital setting and having them readily available in the ed. the logistical difficulties in providing ffp transfusions early in trauma are based on the need for reliable cold storage facilities, specialized transport equipment and personnel to provide them, and a lengthy thawing process. there is also a significant loss of up to 50% due to bag breakage during transport and thawing [35] . ffp is prepared through separation from whole blood and stored at −18 °c or colder with a shelf life of about 1 year. the thawing process requires a 30-37 °c agitated water bath or a warming device cleared by the us food and drug administration; this takes 15-30 min [36] . thawed ffp should be immediately used but can be stored between 1 and 6 °c for up to 5 days. all those factors preclude its immediate availability and delay administration in many settings such as austere environments, the battlefield, and smaller hospitals with limited blood banking capabilities. of great importance and increasing realization is that these same limitations apply in the larger centers as well. the use of plasma in the resuscitation of trauma and hemorrhagic shock began with the work of dr. john elliot, who in 1936 devised a mechanism to separate plasma from red blood cells and store it in a vacuum bottle. he believed plasma was all that was needed to treat hemorrhagic shock [37] [38] [39] . elliot utilized pooling by mixing the plasma of up to eight donors together to neutralize anti-a and anti-b antibodies and eliminate the need for cross-matching [38, 40] . reports of treating shock with dried plasma go back as early as 1938 [41] . in 1940, the british army called upon the american red cross to provide plasma shipped directly to london, and the blood plasma for great britain project started [42, 43] . the "blood for britain" campaign resulted in almost 15,000 units of blood donated from 1940 to 1941 in new york city alone with the majority used to produce liquid plasma, while the rbcs and platelets were discarded. the program was then stopped due to high incidence of bacterial contamination in liquid plasma [42, 43] . meanwhile, dr. max strumia was experimenting with turning elliot's liquid plasma into a sterile powder and refined the drying process by inventing a device for freeze-drying under a vacuum [37, 44] . this was followed by production of several hundred units of dried plasma for testing by the us army and navy. in 1941, freezedried, or lyophilized, plasma was approved for use by the council on pharmacy and chemistry of the american medical association. boxes were designed containing the dried plasma in a bottle accompanied by a bottle of sterile water for reconstitution [37] . lyophilized plasma use started in wwii where millions of units were produced by the american red cross and administered by the us and british armies and also distributed to the allied forces [17, 37, 42] . it was the primary mode of resuscitating combat casualties for most of wwii. the recognition of serum hepatitis as a result of pooled lyophilized plasma transfusion caused it to fall out of favor. it continued to be used in the korean war but was abandoned altogether in the 1950s [37, 42] . the french military continued to produce lyophilized plasma through the mid-1980s when hiv transmission via blood transfusion was recognized. they resumed production in 1994 utilizing small donor pools (under 11 donors) and amotosalen with uv light processing for pathogen reduction [54] . during the same time, the german red cross started processing pooled plasma with a solvent/detergent (s/d) treatment as a method of pathogen inactivation. this continued through the early 2000s when the recognition of possible prion disease transmission caused them to switch to a single-donor approach [42, 45] . production of dried plasma is achieved in two ways: freeze-drying, also known as lyophilization, or spray-drying. lyophilization is achieved by freezing the plasma under a vacuum in a glass container for several days, which decreases the water content to 1-2% [45] . spray-dried plasma production utilizes atomization of liquid plasma via pressurized drying gas to droplets which are then exposed to hot gas (up to 150 °c) in a drying chamber followed by rapid evaporative cooling. this method can dry a unit of plasma (~250 ml) in approximately 25 min [17, 46] . dried plasma can then be reconstituted to its original volume or a concentrated form. multiple pathogen inactivation methods are available to use during the process, and the choice depends on the manufacturer's preference and experience. newer and more accurate viral detection and pathogen inactivation methods have led to the improved safety of blood products, reducing the residual risk of transfusiontransmitted hiv-1 and hcv to approximately 1 in 2 million blood units [47] . pathogen inactivation methods used for plasma include solvent/detergent (s/d) treatment and photochemical inactivation techniques [17] . s/d treatment, which is fda-approved, binds lipid-enveloped viruses and bacteria to inactivate them followed by a filtration process to remove cells and debris, but it has no effect against non-enveloped viruses and prions [48] . rigorous screening standards require testing donors for non-enveloped viruses twice at a 6-month interval to decrease the risk of transmission. moreover, photochemical inactivation utilizes a photosensitizer that binds the dna and rna of pathogens, including non-enveloped viruses, and nucleated cells, followed by ultraviolet light exposure to inactivate them [49] . intercept (cerus corp., concord, ca) is an fda-approved system, which uses amotosalen (a psoralen molecule) to bind dna and rna followed by uv light activation [50] . the mirasol system (terumo bct, lakewood, co), which uses riboflavin as the photosensitizer, is currently approved for clinical use in europe but only approved for investigational use in the usa and canada [51] . methylene blue can also be used with visible light exposure [52] . standard s/d treatment causes a decrease in vwf activity (24%), factor v (37%), protein s (44%), and alpha-2 antiplasmin (79%). similarly, amotosalen + uv light reduces factor vii (23%) and factor viii (27%) [17, 48] . a newer s/d treatment product, octaplas lg (octapharma, lachen, switzerland), received fda clearance in 2013 and employs a prion reduction step and a modified s/d process that better preserves factor levels [17] . reconstituted porcine lyophilized plasma is alkalotic with a ph >8.5, making it highly lethal when injected in swine due to their lack of ability to buffer their plasma [53] . human lyophilized plasma is also alkalotic with a ph near 8; however, it is well tolerated clinically in humans [54] . this increase in ph after lyophilization is attributed to the loss of bicarbonate during the drying process. multiple acidic buffering solutions were studied to evaluate their effect on the hemostatic properties of lyophilized plasma. for example, when ascorbic acid (vitamin c) is added to lyophilized plasma (lp), 84% of the coagulation factor activity was maintained [55] . in a swine model of polytrauma and hemorrhagic shock, a significant decrease in interleukin-6 (il-6) levels was observed in all lp-treated animals compared to those receiving ffp, suggesting an anti-inflammatory effect [55] . this was corroborated by another study using concentrated lp (50%) that showed buffering with ascorbic acid resulted in reduced serum levels of il-6 and tnf [56] . another study examined the effect of other buffers, such as citric acid and hydrochloric acid, on lyophilized plasma. no difference in physiology, coagulation parameters, or blood loss was noted, but animals receiving ascorbic acid had lower il-6 levels and less oxidative dna damage [57] . using higher concentrations of ascorbic acid didn't affect the physiologic benefits of lp, but no improvement in the anti-inflammatory effects or further decrease in dna oxidative damage was detected [53] . the type of fluid used to reconstitute dried plasma has not been shown to affect the degree of inflammation or oxidative dna damage induced by shock in a swine model of polytrauma and hemorrhagic shock [58] . however, the type of fluid used for reconstitution does affect the hemostatic efficacy and ability of lyophilized plasma (lp) to mitigate the effects of shock. animals treated with lp reconstituted with sterile water and lactated ringer's (lr) had less blood loss compared to those reconstituted in normal saline (ns) and hextend. the group that received hextend had persistently elevated inr values and contained the only animal that did not survive the experiment. serum il-6 levels were lowest in the sterile water group when compared to ns [59] . the optimal solution for buffering lyophilized plasma in humans is unknown and will require further investigation. for now, sterile water is used to reconstitute human lp. the process of freezing and thawing plasma is not benign and has several detrimental effects on coagulation proteins. never-frozen liquid plasma was found to have a superior coagulation profile and factor activity, as well as thrombin generation potential, when compared to plasma from thawed ffp [60] . thawed plasma was compared at day 0 and after storage at day 5, and a significant degradation of clotting factors was detected in the older product, with a 40% reduction in thrombin generation potential and significantly decreased hemostatic profile on thrombelastography (teg) analysis [61] . thawed ffp decreased vascular permeability in vitro by a factor of 10; however, that effect decreased to only a factor of 2.5 when 5-dayold thawed plasma was used [62] . these findings reinforce some of the advantages of dried plasma over ffp. concentrated, low-volume reconstitution of lyophilized plasma (50%) has been demonstrated to be safe in a swine polytrauma model of hemorrhagic shock with similar physiologic effects, hemostatic properties, and coagulation parameters (inr and teg) [63] . this could have logistical advantages in packaging and transport on the battlefield as well as in austere environments. the effects of infusing this hypertonic, hyper-oncotic fluid are unknown in humans and will require careful evaluation. spray-dried plasma (sdp) at original concentration was compared with triple concentrated sdp in the resuscitation of a swine model of polytrauma and hemorrhagic shock. in vitro evaluation of coagulation parameters of sdp compared with ffp did not reveal any difference between the two products. however, tripleconcentration sdp showed an increase in clotting factor activity and prolonged pt/ ptt. treatment with all three formulations corrected inr rapidly and increased clot strength (teg-maximum amplitude (ma)). this confirmed that concentrated, lowvolume sdp is as effective as ffp and regular sdp in reversing trauma-associated coagulopathy [64] . the accepted standard for factor loss in frozen then thawed plasma is 25-40% [65] . in vitro analysis of swine ffp vs. lyophilized plasma (lp) coagulation tests (pt, ptt, inr, fibrinogen) did not reveal any statistically significant difference. reconstituted lp has been shown to maintain an average of 86% of coagulation factor activity when compared to ffp [66] . in comparison, spray-drying causes reduction in several factors including 25% for fibrinogen and protein s, 50% for vwf activity, and 70% for factors v and viii. however, this has been shown to have no effect on the ability of sdp to generate thrombin [67] . on the contrary, after a year of storage at −25 °c, lyophilized plasma had no significant change in clotting factors activity when compared to fresh plasma [68] . lyophilized plasma stored as long as 30 years had similar preservation of components [69] . multiple studies demonstrate the safety and efficacy of lyophilized plasma (lp). in a series of studies using a swine model of polytrauma and hemorrhagic shock, lp demonstrated superior hemostatic efficacy to ffp when combined with rbcs in 1:1 ratio. concentrated lp reconstituted to 50% of its volume was also well tolerated and equally effective in correcting shock physiology when compared to unconcentrated lp [56] . in another study, fresh whole blood (fwb), ffp, and lp all corrected coagulopathy equally in a swine model. there was 85% mortality in the crystalloid only group and no mortality in any of the blood products groups [66] . another study showed that when compared with colloid alone, 7-day survival was superior in animals that received spray-dried plasma -this effect was equivalent to that seen in animals that received whole blood [70] . moreover, animals receiving balanced lp-to-rbc had significantly less blood loss than those receiving ffp or lp alone, and lp was as effective as ffp in reversing coagulopathy in this animal model [55] . lyophilized plasma (lp) demonstrated similar effects as ffp, both in vitro and in vivo, on reducing endothelial cell permeability, increasing trans-endothelial resistance, decreasing leukocyte-endothelial binding, and preserving adherens junctions. in an in vitro mouse model of hemorrhagic shock, lp and ffp both equally reduced pulmonary injury, inflammation, and vascular permeability [71] . spray-dried plasma (sdp) also reduced vascular permeability and other indicators of endothelial damage as well as ffp [72] . ffp and sdp equally decreased shock-induced pulmonary vascular permeability in vivo. sdp was also equivalent to ffp in the correction of shock in a mouse model. they both reduced alveolar wall thickening, leukocyte infiltration, and the breakdown of ec junctions [73] . plasma has been shown to have multiple neuroprotective effects in traumatic brain injury. in a swine model of polytrauma, hemorrhagic shock, and tbi, both lyophilized plasma (lp) and ffp were shown to decrease brain lesion size by 50% after 6 h of injury when compared to saline infusion; swelling was also 54% less in plasma-treated groups [74] . a follow-up study to evaluate the long-term effects of resuscitation with ffp vs. lp on neurological outcomes showed similar recovery of cognitive function in studied animals. the brain lesion size was significantly smaller in lp group on experiment day 3, but this effect dissipated by day 10 [75] . another large 30-day animal study recently showed similar neuroprotective results with faster return to baseline neurological function in animals treated with lp and ffp vs. ns [76] . since its reintroduction in the 1990s, lyophilized plasma has been used in a variety of settings around the globe. currently, the largest two manufacturers of lyophilized plasma are the french military and the german red cross. multiple accounts of the use of lyophilized plasma have been reported, including administration at the point of injury, in the ed and in the icu. french lyophilized plasma (flyp) is used by us military special operations under an agreement between the french and us governments as an expanded access investigational new drug application [15, 17, 77] . german lyophilized plasma, known as lyoplas n-w, has been carried by uk foot patrols since 2012. the use of lyoplas n-w was easily integrated into the first responder care package. one case of successful usage by the british military is reported in the literature [78] . the national bioproducts institute in south africa also produces a pooled, s/d-treated, abo-universal lyophilized plasma, bioplasma fdp, which has been in use in south africa since 1996, with a strong record of safety [79] . the norwegian helicopter emergency medical service experience with the use of lyophilized plasma (lyoplas n-w, ab) during a 12-month period reported transfusion of 16 patients having sustained blunt and penetrating trauma, as well as nontraumatic hemorrhage (ruptured aaa, upper gi bleeding, etc.). two patients died on scene, and the remaining were alive at 30 days. no transfusion-related complications were reported. lyophilized plasma is stored at room temperature in the fastresponse car and in the helicopter, making it readily available. pre-transfusion hypotension was seen in 62% of the patients, but only 12% were still hypotensive at the time of admission. median systolic blood pressure increased after prehospital lyophilized plasma transfusion in all patient categories. 68% of the patients received emergency surgery after arrival at the hospital [80] . the swedish armed forces also use lyophilized plasma, and the first civilian helicopter emergency medical systems in sweden started carrying the product in 2015. they published a case report describing a patient with carotid artery injury due to a high-velocity gunshot wound to the neck and in-flight reconstitution and administration of lyophilized plasma (lyoplas n-w) in a medevac helicopter. the reconstitution of lyoplas n-w powder took about 4 min in a dark black hawk helicopter cabin. the hemodynamic stability of the patient improved after administration [81] . the israeli defense force medical corps (idf-mc) introduced lyophilized plasma, in the form of lyoplas n-w, to its protocol of prehospital trauma care and transfusion in 2013 [82] . a case report of its use in a civilian after a motor vehicle accident described their first experience with point-of-injury administration of lyophilized plasma [83] . this was followed by a retrospective review of 109 patients who were transfused with lyophilized plasma from 2013 to 2016. the majority (83%) of patients received only one unit of lyoplas n-w, and only 8.2% received prehospital blood transfusions. there were five instances (4.6%) of difficulty with administration after reconstitution mainly due to low flow rates. side effects were reported in only one female patient who developed chills and shivering during infusion which stopped upon prompt discontinuation [84] . this study is a real-life example of utilizing prehospital lyophilized plasma in early resuscitation of trauma casualties demonstrating safety and feasibility. dr. jean julliard started producing freeze-dried plasma in 1945 after its inception by the us military in wwii. by 1950, the centre de transfusion sanguine des armées (ctsa) became the first european center to produce lyophilized plasma. during the indochina war, almost 40,000 units of lyophilized plasma were delivered to the french military. production was suspended in 1985 due to concerns for hiv transmission. in 1991, production restarted with the first gulf war and has continued since that time [54] . since 1994, french lyophilized plasma (flyp) is made using a pool of less than 11 donors. pooling based on blood type selection allows the dilution and neutralization of natural anti-a and anti-b hemagglutinins, making flyp a universal donor product compatible with any recipient blood type. since 2003, it is also being leukoreduced. starting in 2010, plasma from women with a history of pregnancy is tested for hla antibodies and excluded if positive. that was the same time that flyp started undergoing amotosalen and uv light processing as a pathogen dna/ rna inactivation method. this process was chosen over solvent/detergent treatment due to better preservation of clotting factors. the french hemovigilance program has been monitoring flyp since 1994, and so far, no reactions or infectious complications have been reported out of more than 1100 units transfused [54] . flyp is packaged in glass bottles, shelf-stable in ambient temperatures between 2 and 25 °c for 2 years, and easily rehydrated with 200 ml of water in less than 3 min, allowing for immediate transfusion with rbcs. flyp contains all clotting factors and proteins. after more than 2 years of storage at ambient temperature, the fibrinogen and clotting factor levels of flyp are equivalent to ffp [85] . despite a certain level of factor reduction (20-25%), lyophilization has not been shown to alter in vitro hemostatic efficacy of plasma. when reconstituted, flyp has a ph close to 8 [54] . in 2011, flyp was authorized by the french agency for the sanitary safety of health products (afssaps) for use in civilians in austere settings or until thawed plasma became available [54] . clinical efficacy of flyp was studied in a prospective trial on 87 icu patients in afghanistan and was found to be safe and efficacious in the management of polytrauma and shock [86] . furthermore, the difference in administration times between flyp and ffp in a level 1 trauma center was studied. retrospective analysis showed significantly less time to product administration between patients receiving flyp vs. ffp (median 15 vs. 95 min). this is consistent with similar reports in the literature of time to ffp transfusion [21] . subsequently, time to achieve 1:1 resuscitation ratio with rbcs was shorter in flyp group. there were also significantly less cases of massive transfusion utilization and rbc transfusion in the flyp group compared to the ffp group (7 vs. 45%). no differences in hospital length of stay, icu length of stay, or 24-h mortality between the two groups were noted [87] . recently, a randomized open-label clinical trial of 48 patients who were assigned to receive 4 units of flyp or ffp within 6 h of injury was completed. patients in the flyp group demonstrated less time from randomization to infusion compared to those in the ffp group (median 14 min vs. 77 min). this led to higher levels of fibrinogen achieved within 45 min of randomization, as well as a greater improvement in inr, factor v, and factor ii levels. the difference in coagulation parameters between the two groups remained significant at 6 h. however, there was no difference detected in mortality between the two groups [88] . in the 1990s, the german red cross blood service west produced solvent/detergent (s/d) treated lyophilized plasma using pooled plasma. due to concern for creutzfeldt-jakob-type prion disease transmission, which is not inactivated by standard s/d treatment, pooled plasma was replaced with single-donor lyophilized plasma in 2007 [45] . this product is licensed under the name lyoplas n-w (german red cross blood service west, hagen, germany). quarantined plasma from a single donor is stored frozen for at least 4 months until the donor returns for retesting for hiv, hepatitis c virus, hepatitis b virus, hepatitis a virus, and parvovirus b19. after the quarantine, the plasma is thawed and connected by sterile docking to the patented steam-sterilized "bottle-in-bag" system, which consists of a glass bottle and a rubber stopper inside a plastic bag. during transfer into the glass bottle, the plasma passes through a filter with a nominal pore size of 0.2 μm. once 200 ml of plasma is transferred, the bottle is closed with the stopper and removed from the system. plasma is then frozen to −30 °c followed by lyophilization in specially designed freeze-dryers. the lyophilization is accomplished by a stepwise increase of the temperature from −45 °c to +15 °c, resulting in water content below 1% [45] . sterile water for reconstitution (200 ml) is included in the lyoplas n-w kit and accomplished within 10 min depending on the plasma composition and water temperature. transfusion can be accomplished via the glass bottle or the plastic bag, which allows for pressure infusion if needed. after storage at 2-8 °c for 24 months, lyoplas n-w only had a 10% reduction in factor v, viii, and vwf. all other factors remained stable. storage at room temperature, however, led to 54% decrease in fibrinogen levels and vwf activity. this is why the shelf life of lyoplas n-w has been restricted to 15 months only. after reconstitutions, factor degradation increases over time at room temperature. at 48 h, factor viii and protein s levels decreased by 66 and 50%, respectively. only 10% degradation of those factors was noted in the first 6 h, however. this is why it is recommended to use lyoplas n-w within 6 h of reconstitution [45] . the us department of defense (dod) and the biomedical advanced research and development authority (barda) are sponsoring multiple different programs to provide dried plasma products in different forms (lyophilized and spray-dried). the aim is to make the distribution, storage, and administration of plasma in combat and civilian environments safe and feasible [79] . historically, there was a dried plasma product licensed in the usa under plas-sd manufactured by vitex (melville, ny) which was required to have a black box warning due to the risk of adverse thromboembolic events caused by low levels of protein s. this was attributed to the solvent/detergent treatment. however, newer technology provided by octaplas lg seems to have resolved the problem [79] . the vitex product is no longer available. hemcon medical technologies, inc. (portland, oregon) was in the process of developing a dried plasma product for the us army medical research and materiel command (fort detrick, md) between 2008 and 2013. it was going to be a singledonor lyophilized plasma product derived from licensed ffp. in 2011, the product underwent a successful phase i clinical trial and was shown to have factors within the normal range [17, 89] . unfortunately, the partnership ended in 2014 due to business reasons. agency program since 2008. s/d treatment using a process licensed from octapharma (lachen, switzerland) that is effective against lipid-enveloped viruses and other pathogens is utilized. the s/d process also removes immunogenic lipids, and a filtration step removes cellular debris and proinflammatory microparticles. phase i clinical trials were completed in 2016, and developers have a goal of being licensed by 2020 [17, 92, 93] . nova laboratories (leicester, uk) will perform the spray-drying and packaging for the product [79] . velico medical (beverly, ma), is developing a spray-drying device and proprietary bag system (frontline odp) that will enable blood banks to produce licensed, single-donor, spray-dried plasma units locally within 30 min. this program is conducted under a contract from barda, a part of the us department of health and human services, and is still in the preclinical phase [46, [93] [94] [95] . this product will provide a certain independence from manufacturers and allow local augmentation of production in times of need [79] . damage control resuscitation is now the standard of care in the treatment of hemorrhagic shock. it is clear that trauma patients with serious injury will benefit from dcr within minutes of injury. plasma transfusion is an integral part of this concept but suffers from several logistical constraints. this also makes it an area where significant advancements are necessary and can improve patient outcomes. early delivery of plasma is one avenue that seems to suffer the most. many challenges exist that hinder this goal including physical requirements for storing and transporting ffp, required personnel, and thawing times. certain steps taken by major trauma centers to remedy that have been successful, including having thawed plasma ready in the ed at all times and carrying thawed and liquid plasma in the prehospital setting. these measures are costly and require a large-scale operation. smaller hospitals will not be able to accommodate such measures. patients presenting to such facilities will suffer worse outcomes from delay in administration of plasma until it is available or until transported to a larger center. physicians are forced to use crystalloid and colloid in such situations to stabilize patients. furthermore, trauma casualties in remote locations requiring long transport times or austere environments requiring prolonged extrication will be at a huge disadvantage. finally, soldiers and combat casualties are also negatively affected by the delay of plasma transfusion, which has been demonstrated over and over again. the solution is to have a product that is readily available, easy to store and transport, and can be administered quickly and safely. dried plasma provides all these advantages. it can be stored up to 2 years at room temperature and reconstituted within minutes. it's been shown to be safe and efficacious clinically and in animal models with similar coagulation properties to ffp. a dried plasma product introduced in the usa will allow for earlier plasma administration starting prehospital and continuing into the hospital setting, which will likely improve patient outcomes, as demonstrated by the french experience [87] . dried plasma should certainly replace the recommended crystalloid administration in the atls guidelines, which were designed to accommodate all levels of practice and take into account the variable availability of blood products. taking this concept one step further, it may prove beneficial and efficient to replace ffp altogether, especially when time to transfusion is a critical element of care, thus eliminating the need for cold storage facilities and complicated thawing equipment and procedures. the future of lyophilized plasma is exciting, and while it is an old product, it will likely see a new beginning. transport time and preoperating room hemostatic interventions are important time is the enemy: mortality in trauma patients with hemorrhage from torso injury occurs long before the "golden hour the prospective, observational, multicenter, major trauma transfusion [prommtt] study damage control resuscitation: directly addressing the early coagulopathy of trauma major abdominal vascular trauma-a unified approach immediate versus delayed fluid resuscitation for hypotensive patients with penetrating torso injuries increased plasma and platelet to red blood cell ratios improves outcome in 466 massively transfused civilian trauma patients preparation of battle casualties for surgery acute traumatic coagulopathy early coagulopathy predicts mortality in trauma the prevalence of abnormal results of conventional coagulation tests on admission to a trauma center early coagulopathy in multiple injury: an analysis from the german trauma registry on 8724 patients ten-year analysis of transfusion in operation iraqi freedom and operation enduring freedom optimal trauma resuscitation with plasma as the primary resuscitative fluid: the surgeon's perspective the ratio of blood products transfused affects mortality in patients receiving massive transfusions at a combat support hospital dried plasma: state of the science and recent developments constant challenges and evolution of us military transfusion medicine and blood operations in combat impact of policy change on us army combat transfusion practices transfusion of plasma, platelets, and red blood cells in a 1:1:1 vs a 1:1:2 ratio and mortality in patients with severe trauma: the proppr randomized clinical trial an emergency department thawed plasma protocol for severely injured patients association of prehospital blood product transfusion during medical evacuation of combat casualties in afghanistan with acute and 30-day survival prehospital transfusion of plasma and red blood cells in trauma patients effects of fresh frozen plasma, ringer's acetate and albumin on plasma volume and on circulating glycocalyx components following haemorrhagic shock in rats syndecan-1 restitution by plasma after hemorrhagic shock fresh frozen plasma lessens pulmonary endothelial inflammation and hyperpermeability after hemorrhagic shock and is associated with loss of syndecan 1 plasma restoration of endothelial glycocalyx in a rodent model of hemorrhagic shock early resuscitation with fresh frozen plasma for traumatic brain injury combined with hemorrhagic shock improves neurologic recovery differential effects of fresh frozen plasma and normal saline on secondary brain damage in a large animal model of polytrauma, hemorrhage and traumatic brain injury traumatic brain injury and hemorrhagic shock: evaluation of different resuscitation strategies in a large animal model of combined insults fresh frozen plasma resuscitation provides neuroprotection compared to normal saline in a large animal model of traumatic brain injury and polytrauma early plasma transfusion is associated with improved survival after isolated traumatic brain injury in patients with multifocal intracranial hemorrhage advances in military, field, and austere transfusion medicine in the last decade fresh whole blood use by forward surgical teams in afghanistan is associated with improved survival compared to component therapy without platelets damage control resuscitation is associated with a reduction in resuscitation volumes and improvement in survival in 390 damage control laparotomy patients physical and thermal properties of blood storage bags: implications for shipping frozen components on dry ice freeze-dried plasma the plasma wars: a history a preliminary report of a new method of blood transfusion a report on the use of a perfected evacuated unit for blood transfusion office of the surgeon general, dept. of the army a study of experimental and clinical shock with special reference to its treatment by the intravenous injection of preserved plasma plasma transfusion: history, current realities, and novel improvements the blood plasma for great britain project the intravenous use of serum and plasma, fresh and preserved quality of freeze-dried [lyophilized] quarantined single-donor plasma spray: single-donor plasma product for room temperature storage detection of hiv-1 and hcv infections among antibody-negative blood donors by nucleic acid-amplification testing solvent/detergent plasma: pharmaceutical characteristics and clinical experience fundamentals of the psoralen-based helinx technology for inactivation of infectious pathogens and leukocytes in platelets and plasma pharmacokinetic study of ffp photochemically treated with amotosalen (s-59) and uv light compared to ffp in healthy volunteers anticoagulated with warfarin inactivation of viruses in platelet and plasma products using a riboflavin-and-uv-based photochemical treatment a comparison of methods of pathogen inactivation of ffp effect of ascorbic acid concentrations on hemodynamics and inflammation following lyophilized plasma transfusion the evolving role of lyophilized plasma in remote damage control resuscitation in the french armed forces health service lyophilized plasma for resuscitation in a swine model of severe injury the use of lyophilized plasma in a severe multi-injury pig model lyophilized plasma reconstituted with ascorbic acid suppresses inflammation and oxidative dna damage reconstitution fluid type does not affect pulmonary inflammation or dna damage following infusion of lyophilized plasma comparison of the hemostatic efficacy of low-volume lyophilized plasma reconstituted using sterile water, lactated ringer's, normal saline, and hextend solutions better hemostatic profiles of never-frozen liquid plasma compared with thawed fresh frozen plasma multiple levels of degradation diminish hemostatic potential of thawed plasma protective effects of fresh frozen plasma on vascular endothelial permeability, coagulation, and resuscitation after hemorrhagic shock are time dependent and diminish between days 0 and 5 after thaw hyperosmolar reconstituted lyophilized plasma is an effective low-volume hemostatic resuscitation fluid for trauma development and testing of low-volume hyperoncotic, hyperosmotic spray-dried plasma for the treatment of trauma-associated coagulopathy quality of therapeutic plasma-requirements for marketing authorization development and testing of freeze-dried plasma for the treatment of trauma-associated coagulopathy all plasma products are not created equal lyophilized human reference plasma for coagulation factors: evidence for stability of factors i, ii, v, and vii through xii chemical analysis of a 30-year-old bottle of lyophilized plasma hemostatic and pharmacologic resuscitation: results of a long-term survival study in a swine polytrauma model lyophilized plasma attenuates vascular permeability, inflammation and lung injury in hemorrhagic shock spray-dried plasma and fresh frozen plasma modulate permeability and inflammation in vitro in vascular endothelial cells fresh frozen plasma and spray-dried plasma mitigate pulmonary vascular permeability and inflammation in hemorrhagic shock early treatment with lyophilized plasma protects the brain in a large animal model of combined traumatic brain injury and hemorrhagic shock early resuscitation with lyophilized plasma provides equal neuroprotection compared with fresh frozen plasma in a large animal survival model of traumatic brain injury and hemorrhagic shock resuscitation with lyophilized plasma is safe and improves neurological recovery in a long-term survival model of swine subjected to traumatic brain injury, hemorrhagic shock, and polytrauma dod advances development of freeze-dried plasma for battlefield deployment: u.s. medicine freedom from frozen: the first british military use of lyophilised plasma in forward resuscitation comprehensive us government program for dried plasma development freeze dried plasma and fresh red blood cells for civilian prehospital hemorrhagic shock resuscitation helicopter in-flight resuscitation with freeze-dried plasma of a patient with a high-velocity gunshot wound to the neck in afghanistan -a case report freeze-dried plasma at the point of injury: from concept to doctrine pointof-injury use of reconstituted freeze-dried plasma as a resuscitative fluid: a special report for prehospital trauma care prehospital administration of freeze-dried plasma, is it the solution for trauma casualties? freeze dried plasma: a french army specialty use of freeze-dried plasma in french intensive care unit in afghanistan use of french lyophilized plasma transfusion in severe trauma patients is associated with an early plasma transfusion and early transfusion ratio improvement french lyophilized plasma versus fresh frozen plasma for the initial management of trauma-induced coagulopathy: a randomized open-label trial the safety of autologous lyophilized plasma versus fresh frozen plasma in healthy volunteers -full text view -clinicaltrials.gov ascending doses of autologous fdp vs ffp -full text view -clinicaltrials.gov tfx] announces commencement of phase i clinical study [fdp-1] of replas™ freeze-dried plasma | teleflex incorporated resusix™ fresh frozen plasma [ffp] hemostatic volume resuscitation therapeutic | entegrion safety study of spray-dried solvent/detergent-treated plasma for infusion in healthy volunteers -full text view -clinicaltrials.gov velico medical announces barda exercise of $15.5 million contract option -velico medical velico medical raises another $19m for frontline odp spray-dried plasma -massdevice key: cord-006192-bqwchhwk authors: persson, carl g. a. title: plasma exudation and asthma date: 1988 journal: lung doi: 10.1007/bf02714025 sha: doc_id: 6192 cord_uid: bqwchhwk several pieces of evidence support the view that exudation of plasma into the airway wall and into the airway lumen occurs in asthma. vascular leakage of plasma results from inflammatory mediator-induced separation of endothelial cells in postcapillary venules belonging to the tracheobronchial circulation. whereas proposed mediators of asthma induce reversible leakage, several antiasthma drugs exhibit antileakage effects in animals and humans. potential consequences of plasma exudation are many. mucosal/submucosal edema might contribute to airway hyperresponsiveness. plasma exudate in the airway lumen in asthma may contribute to sloughing of epithelium, impairment of mucociliary transport, narrowing of small airways, and mucus plug formation. exuded plasma may cause airway inflammation and constriction because of its content of powerful mediators, and chemoattractant factors and plasma proteins may condition the inflammatory cells abundant in asthmatic airways to release mediators in response to stimuli that otherwise would be innocuous to the cells. it is concluded that inflammatory stimulus-induced increase in macromolecular permeability of the tracheobronchial microvasculature and mucosa may be a significant pathogenetic mechanism in asthma and that the postcapillary venular endothelium and airway epithelium that regulate leakage of plasma are important effector cells in this disease. vascular leakage of plasma is a major sign of inflammation. this factor may deserve attention in asthma, a disease that has an important inflammatory component. however, the tracheobronchial venular endothelium, which regulates airway inflammatory plasma leakage, has not been considered an important effector cell of the lung. the occurrence of plasma leakage is nevertheless supported by findings of large amounts of plasma proteins in the sputum, mucus plugs, and in specific airway lavage fluid obtained from asthmatics [18, 38, 52, 55, 61, 75, 123] . in addition, it has been observed in experimental animals that exposure of tracheobronchial mucosa to inflammatory mediators causes a rapid movement of large plasma solutes not only into the airway wall but also into the lumen [43, 111, 115] . the physical and inflammatory effects of the plasma exudate and its content of protein-derived mediators would have a primary role in airway defense. plasma leakage in airways may be linked with many facets of the pathophysiology of airway diseases such as asthma and rhinitis [38, 105] . vascular leakage of large molecules is an active process under physiological and pharmacologic control [24, 64, 78, 89, 102, 116, 134, 145, 153] . this leakage is generally referred to as increased vascular permeability. this review discusses mechanisms and potential consequences of increased tracheobronchial microvascular permeability. tracheal and bronchial arteries carrying systemic blood nourish the walls of the airways and their accompanying nerves and vessels. species differences and variations within species exist for the origin and distribution of these arteries. capillary and precapillary anastomosis between the bronchial and pulmonary circulation has been demonstrated and there are different views as to how far the bronchial arteries travel in peripheral airways, but terminal bronchioles and alveoli may be supplied by these vessels [9, 31, 81, 90, 109] . the bronchial circulation normally receives about 1% of the cardiac output [9] and a large fraction of bronchial blood flow may go to the airway mucosa/submucosa [103] . the bronchial veins from the first 2-3 generations of bronchi drain into the azygous veins and then into the right heart. the remaining bronchial blood drains into pulmonary capillaries and veins and enters the left heart [31, 90] . different workers have demonstrated the presence of an extensive microvasculature in the trachea and bronchi [72, 90, 100, 132] (fig. 1) . on either side of the bronchial muscle layer there is an abundance of capillary-venular plexuses coupled to a relatively sparse arterial supply. both in the larger and the smaller bronchi the plexuses occupy the entire surface of the submucosal layer [72] . a rich continuous subepithelial network of microvessels would regulate clearance, and possibly distribution (from large to small airways), of inflammatory mediators and inhaled drugs [70, 72] . the profusion of the microvascular network of the airways may be illustrated by isolating the lung and perfusing it only through the pulmonary artery at normal pressures. the microvasculature of airways including the trachea will immediately be quite well perfused also, and this must have been accomplished by a retrograde microvascular flow along the airways [70] . the superficial bronchial microcirculation also has a role in the temperature and moisture conditioning of inhaled air [10, 132] . this function may be of particular relevance to asthmatic subjects who may respond with plasma exudation to inhalation of cold and dry air [107, 109] . a role for plasma exudation in "dry-air-induced asthma" may be hypothesized for 2 main reasons: (1) in inflamed airways it is vessel fluid that humidifies incoming air whereas other sources are used under normal conditions [25, 45] ; (2) effective protection against this nonpharmacologic provocation is provided by drugs such as cromoglycate and glucocorticosteroids, which may have potent antileakage effects at airway endothelial-epithelial barriers [42, 107] . alterations in bronchial blood flow will affect the delivery of plasma and white cells, the perfused surface area, and the microvascular hydrostatic pressure. the flow may be highl.y increased through tissue that is affected by inflamma-tion [8] and the hydrostatic pressure increases in venules, from which exudation takes place in local hyperemia [153] . the hydrostatic microvascular pressure is dependent not only on the arterial and venous pressures but also on the ratio of post-to premicrovascular resistance. vasodilation usually increase this ratio [82] and thus promotes plasma exudation. it has long been recognized that once permeability is increased changes in blood flow may determine the degree of plasma exudation [64, 153] . however, pronounced synergistic effects as demonstrated in the skin between flow-increasing and permeability-increasing mediators [147] may not occur in airway mucosa/submucosa that has a high basal perfusion. the neural, hormonal, and pharmacologic regulation of tracheobronchial blood flow has many of the general characteristics of a systemic circulation [9, 73, 103, 109, 112] . many agents including histamine, bradykinin, acetylcholine, substance p, vip, and prostaglandins have been demonstrated to increase tracheobronchial blood flow [9, 73, 107] . under physiological conditions fluid equilibrium is maintained by a balance between the hydrostatic pressure in the capillary bed, which tends to drive fluid out of the vascular compartment, and a counteracting force of the osmotic pressure of plasma proteins. inflammation brings about dramatic changes in the transmural colloid osmotic pressure gradient. after excluding a number of factors (changes in the blood, increased microvascular pressure, changes in the surrounding tissue, etc.), julius cohnheim [24] , more than 100 years ago, concluded that the inflammatory extravasation of protein-rich fluid must be due to noxious stimuli acting directly on the microvascular wall to cause a molecular change resulting in increased permeability. cohnheim reported that the inflammatory exudate is concentrated and that this is due largely to its proteinaceous nature (differing from high-pressure edema fluid that is protein-poor) [24] . somewhat reluctantly he could then make his reasoning fit with previous publications by julius arnold, who had shown that injected dyes always passed through the vessel wall between endothelial cells [7, 24] . more recent ultrastructural, pathophysiological, and pharmacologic studies of systemic microvascular beds have shown that inflammatory mediatorinduced leakage of protein-rich plasma occurs in postcapillary venules (diameter 10-50 ~m) through large gaps (up to 1 tzm) between endothelial cells [23, 62, 64, 78, 89, 116, 122, 134, 145, 153] (fig. 2 ). in the delayed inflammatory response to mild thermal burns and some other types of injurious stimuli an inflammatory exudate may come both from capillaries and venules [27, 64, 87] , but no mediator has yet been demonstrated to produce capillary leakage [64, 87] . the target cells for inflammatory mediators have thus been identified as venular endothelial cells. plasma escapes through the mediator-induced interendothelial gaps and filters through the endothelial basement membrane, which offers little hindrance to diffusion of plasma proteins [153] . the concentration ratios of different proteins may be similar in blood plasma and in inflammatory exudate, indicating that there is a bulk flow of proteinaceous plasma out of leaky postcapillary venules [8, 12, 59] . contraction of venular endothelial cells has been a favored mechanism to explain mediator-induced macromolecular leakage [78] . this possibility is supported by the presence of actomyosin [13] and bundles of fibrils that could form an endothelial contractile machinery [64] . as in smooth muscle, endothelial contractility may be calcium-dependent. the contraction hypothesis is attractive because, as a corollary, the pronounced ability of endothelial cells to close mediator-induced gaps could be explained as a relaxation of these cells. another view on the mediator-induced deformation of endothelial cells has been discussed by zweifach [152] . inflammatory leaks may be produced by effects on elements interlocking endothelial cells. a weakening of these forces may change endothelial cell shape and cause increased permeability. the attachment of these cells to the basement membrane is reported to be particularly loose in collecting venules [152] , which would facilitate deformation there. also the surface material of the endothelial cells may be involved in macromolecular permeability [126] . at sites of inflammation there may also be changes in the configuration of the filamentous gel making up the basement membrane, and the ground substance may be transformed from a gel to a sol state (shown by rapid dispersion of an injected colloid, which otherwise forms a distinct bleb only) [153] . zweifach [153] emphasized that in chronic diseases venules may be particularly sensitive due to defects in the collagenous and reticular fibers of the perivascular tissue that, together with the basement membrane, provide mechanical support for the vessel. since plasma exudation in theory can be causally linked with several facets of asthma pathology, it would be of interest to examine whether such differences exist between normal and asthmatic airway microvessels. recent ultrastructural examination of biopsies by laitinen and laitinen [71] has demonstrated that subepithelial postcapillary venules have endothelial gaps in asthmatic but not in normal airways. new mediators are continually being discovered and characterized as factors of potential importance in asthma and other inflammatory diseases [5] . irrespective of the chemical class of the mediator and whether it is applied extra-or intravascularly, the histologic and ultrastructural characteristics of the induced acute macromolecular leakage appear identical [14, 64, 78, 1t6, 136] . however, different vascular beds may differ in their sensitivity to individual mediators. not only are pulmonary microvessels quite resistant to histamine-type mediators [63, 64, 79, 114] but also some systemic beds such as microvessels of rat intestinal mucosa may be resistant [64] . the tracheobronchial microvasculature has generally responded in a sensitive way to leakage-inducing mediators [e.g., 109, 113] . many proposed mediators of asthma are capable of inducing both bronchoconstriction and vascular leakage but may also differ in these 2 effects. muscarinics are potent constrictors of airway smooth muscle but are without or almost without effects on microvascular permeability to macromolecules [112] . histamine produces leakage in cat tracheal microvessels [41] although it may relax rather than contract cat large airways [3, 110] . similarly, paf-acether is a poor constrictor of guinea pig trachea but is effective in producing plasma exudate in this tissue [111, 115] . due to their effect on vascular permeability, inflammatory mediators may in part produce bronchoconstriction through smooth muscle plasma-derived peptides. the acute allergen-induced response in guinea pigs may be associated with airway edema [32] . although edema could not be demonstrated, vascular leakage of macromolecules into the airway wall and lumen was pronounced in an acute ige-driven anaphylactic reaction in the tracheal mucosa of anaesthetized guinea pigs [111, 113] . extensive deposits of fibrin that would be secondary to a vascular leak were the most striking characteristic of ige-dependent late phase reactions in human skin, whereas cellular infiltration was not a consistent finding [128] . chemical sensitizers such as plicatic acid and isocyanates (e.g., toluenediisocyanate [tdi]) are important inducers of occupational asthma. bronchial provocation with tdi causing both immediate and late phase responses is associated with significantly increased levels of albumin in bronchoalveolar lavage fluid [43] . tdi is also a potent inducer of very prolonged (>24 h) vascular and epithelial macromolecular permeability in guinea pig trachea (erjefo_lt and persson, unpublished data). airway bronchial lavage in patients with asthma due to exposure to western red cedar (plicatic acid) showed a 10-fold higher albumin concentration in the lavage fluid than in normal subjects [75] . the lavage was performed 24-48 h after provocation, when symptoms had subsided. preliminary observations with allergen and chemical sensitizers are thus compatible with the possibility that plasma exudate may contribute to latephase and sustained airway reactions. leakage of macromolecules is evident within 10ths of seconds after application of a directly acting inflammatory agent on systemic microvascular beds (including the tracheobronchial circulation). a maximum effect of mediators such as histamine, bradykinin, tachykinins, and leukotrienes is usually established within 3-•0 rain [14, 84, 89, 111, 112, 116, 134, 136, 145, 151] . the response then declines quickly and normal low permeability is restored within a few minutes up to half an hour. during some time after the development of a response the affected tissue is partly refractory to further permeability effects [11, 20, 51, 137] . tachyphylaxis or refractoriness is not absolute. prolonged vascular leakiness may be due to sequential effects of different mediators or intermittent release of mediators that avoid tachyphylaxis. since bradykinin did not exhibit tachyphylaxis in human skin responses [i 1, 51], it may participate in delayed inflammatory reactions [11] . menkin [85] demonstrated that a cell-free plasma exudate injected into rabbit skin produced prominent vascular leakage of macromolecules. due to their distribution and activity, plasmaderived mediators may exert positive feedback effects on the venular wall and be responsible in part for maintaining high vascular permeability. the inflammatory breakdown of blood and tissue proteins will not only produce mediators but also increase the number of molecules and, hence, increase interstitial osmotic pressure, thus promoting transudation [44] . miles and co-workers [20, 29, 89] demonstrated interesting biphasic as well as sustained permeability responses to bacterial toxins injected into guinea pig skin. hence many types of injury may cause an immediate phase of leakiness that often is short-lasting; after 1-2 h a late phase of increased microvascular permeability follows that is much more sustained [145] . we have recently observed that topical application of paf-acether on guinea pig tracheal mucosa in vivo produced both an acute [11 i] and a late phase vascular leakiness 5 h after provocation [115] , which has not as yet been seen with other mediators. sticking of white cells to endothelium and their subsequent migration across the vascular wall characterize most inflammatory processes. as with protein leakage the leukocyte-endothelium interactions occur in postcapillary venules and the diapedesis is through endothelial intercellular junctions [64, 80, 134] . however, inflammatory extravasation of leukocytes and macromolecular solutes is induced via different mechanisms and can occur separately: white cells apparently have a protein-tight seal during migration, and mediator-induced leakage of plasma can occur without any cellular escape [44, 50, 64, 153] . tissue leukocytosis may be associated with delayed responses and a causal relationship between leukocytes and leakage has been suggested [142] . however, neutropenia may not suppress the initial or the delayed permeability response [145, 148, 149] , and mediators such as anaphylatoxins and paf-acether, which have been proposed to act through polymorphonuctear leukocytes, may have pronounced vascular leakage effects independent of leukocytes and platelets [14, 119] . although participation of leukocytes is likely, their suggested pivotal role [142] in the development of a sustained microvascular permeability in inflammation has not been proven. the mediators may come from many sources, the stationary and migrating inflammatory cells being the most widely explored. airway epithelial ceils may produce arachidonate mediators [97] and vascular endothelial cells may also generate several of the inflammatory mediators, including paf acether and arachidonate products [e.g., 19] . neuropeptides have been proposed as mediators of vascular permeability but substance p and other tachykinins may not be as effective as inflammatory agents in human airways as they are in the guinea pig [112] . of particular relevance for the present discussion (i.e., the sequelae of microvascular leakage) must be the preformed and dormant protein mediators circulating in the bloodstream. huber and koessler [61] included in their review the information that the serum of 1 patient dying of asthma "was very toxic for animals, 0.05 c.c. causing death of a guinea pig." circulating kinins [1] and esterase activity [16] may be elevated and kininogen decreased [16] in asthma. ciliary dyskinetic factors have been detected in asthmatic serum [150] as have indices for activation of complement [86] . hence, the plasma of asthmatic subjects may be particularly noxious. the plasma proteins leaking through the venular gaps may be activated by negative surface charges, proteases, and other factors during their transvascular passage and upon arrival in inflamed tissue [12, 48, 87, 88, 94, 120, 133, 141, 144] . both in the airway wall and lumen proteins of the kinin, complement, clotting, and fibrinolysis systems may generate a variety of inflammatory, bronchoconstrictory, and chemoattractant mediators. bradykinin, which is a powerful provocateur in asthma [56, 130] , is but 1 of a plethora of plasma-derived mediators. gerberick et al. [47] showed that rabbit alveolar macrophages were unable to release reactive oxygen intermediates unless they were conditioned by prolonged presence of plasma proteins. exuded plasma may thus, by direct actions on a variety of target cells and by recruitment and conditioning of inflammatory cells, be an amplifying factor that escalates and sustains the inflammatory process in asthmatic airways [105] . the result of plasma leakage is generally thought of as edema, and edema is accepted as a characteristic sign of asthmatic airways [38, 58, 65, 124] . this view agrees with general descriptions of inflammation of mucosal membranes and is supported by histologic preparations that show "edema spaces" in airways obtained from patients dying of asthma [38, 58] . however, many workers who have done postmortem or biopsy examinations have not reported or been able to identify edema in asthmatic airways [4, 22, 30, 57, 60, 61, 85, 125, 139, 146] . changes such as enlarged bronchial glands and increased thickness of the epithelial basement membrane and smooth muscle layer have been well documented [30, 39, 139] , whereas edematous changes have not been easy to quantify. the relative lack of data on edema may be explained in part by movement of plasma exudate into the airway lumen. this possibility is supported by the abundant occurrence of plasma proteins in asthmatic airways [18, 38, 52, 55, 61, 75, 123] and by observations in experimental animals. inflammatory mediators such as bradykinin, histamine, leukotrienes, tachykinins, paf-acether, and allergen applied to the tracheal mucosa of anesthetized guinea pigs produced acute extravasation of plasma and tracer macromolecules. edema could not be identified but extravasated large solutes were recovered in tracheal luminal fluid within a few minutes after provocation [43, [111] [112] [113] 115] . in contrast to tracheobronchial venules, the pulmonary microvessels are resistant to histamine-type mediators [63, 64, 79, 114] . this aspect, together with abundant microvascular connections between the bronchopulmonary circulations, has stimulated a debate as to the relative importance of bronchial microvessels to fluid and protein exchange in pulmonary inflammation. in adult respiratory distress syndrome (ards) pulmonary edema and increased permeability to macromolecules in the pulmonary vessels and the alveolar wall are present [59] , but bronchial venules may also leak plasma [117] . wheezing is one of the symptoms of ards, and, as in bronchial asthma, survivors of ards may exhibit increased airway responsiveness to methacholine inhalation challenges [i 31]. hence, it cannot be excluded that bronchovascular plasma exudation is one of the factors contributing to asthmalike symptoms in pulmonary inflammatory diseases. it has been calculated that small changes in mucosal thickness could have a profound influence on the tendency to airways closure as well as explain airway hyperresponsiveness to bronchoconstricting agents [65] . slight edema of tissues between the bronchial muscle and the epithelium would thus only marginally reduce the baseline caliber of the airway lumen and be difficult to detect, but could cause abnormally large increases in resistance to airflow during bronchoconstriction. it has not yet been studied whether airway edema, similar to pulmonary edema [26, 66, 127] , may lower the threshold for sensory receptor stimulation. in 1900, fraenkel [46] suggested that extensive epithelial shedding is a distinguishing characteristic of asthmatic airways. this proposal has been repeatedly substantiated [e.g., 37, 91] and dunnill [37] has suggested that mucosal edema and transepithelial passage of plasma exudate cause the shedding of epithelium. however, a significant volume of proteinaceous plasma can rapidly traverse the epithelial barrier without causing shedding [i 15] . sustained inflam-mation and effects of potent and toxic cellular mediators such as eosinophilderived proteins [49] may be required for significant shedding to occur. as discussed above several additional consequences of plasma exudation in the airways relate to the presence of plasma protein-derived mediators in the exudate that are capable of producing bronchoconstriction and inflammation. albumin is a normal constituent of tracheobronchial luminal liquid [17, 75, 104, 111] . stockley et al. [135] determined the relation between sputum/serum concentration ratios and stokes radius for 5 selected proteins in chronic bronchitis. during stable noninfected conditions there was a significant negative correlation between the ratio and the protein size consistent with a passive diffusion of these proteins [135] . during infection the ratio was increased more than 10-fold [21] . this finding tallies with observations in upper airways: nasal washings obtained during viral rhinitis contain much elevated levels of serum proteins and kinin activity [6, 33, 95, 121] . also in many noninfectious types of actue inflammation the epithelial permeability to macromolecules may increase dramatically. large amounts of charged macromolecules such as albumin and uncharged fluorescein-labeled dextran (mw 150,000 daltons) traversed vascular and epithelial barriers of guinea pig tracheas superfused with mediators or challenged with allergen [43, 111, 115] . this highly increased permeability to inflammatory stimuli reversed spontaneously and was significantly prevented by drugs [42, 43] . a pinocytotic transport mechanism would not suffice to bring about such a sudden transepithelial passage of a large volume of plasma [17, 78] . perhaps intercellular junctions of tracheobronchial epithelium can be opened for macromolecular passage as in alveolar epithelium in high-permeability pulmonary edema [28, 59, 99] . an ultrastructural study of inflamed guinea pig trachea has demonstrated that intraluminal horseradish peroxidase penetrates the wall between epithelial ceils [129] . a series of observations of nasal liquid composition in atopic subjects challenged with allergen agrees with the notion that inflammatory stimuli induce a rapid, transient bulk flow of plasma macromolecules (and activation of peptide mediators) extravascularly and across the nasal epithelial lining [12, 120] . it is of great interest that antiasthma drugs, notably glucocorticoids and xanthines, reduce this plasma leakage [96, 108, 118] . mediator-induced increase in the permeability of the epithelial barrier can obviously be as dramatic as that across the venular endothelial lining. experiments indicate that the inflammatory plasma leakage does not require tissue destruction. instead, epithelial permeability to large molecules may be considered an active, reversible process that is under the control of mediators, hormones, and drugs. the subcellular~epithelial mechanisms involved in these permeability changes and details of intercellular pathways for leaking macromolecules remain unexplored. in recent years, airway mucosal or epithelial "permeability" has received widespread attention as a pathogenetic factor. what is usually measured in studies of this kind of "permeability," in particular in human subjects, is the rate of transfer of inhaled 99mtc-labeled diethylenetriamine penta-acetate (dtpa) into the blood. dtpa is a relatively small molecule (mw 492 daltons) and its passage across airway epithelial-endothelial barriers may be regulated by mechanisms entirely different from those involved in leakage of large plasma proteins. hence neither vascular nor mucosal permeability to plasma macromolecules may be reflected in studies using dtpa. furthermore, lung clearance of inhaled dtpa may largely be across alveolar barriers. respiratory mucosal permeability determined with dtpa was not increased in asthma [40] and, when it was increased, as in smokers, this did not correspond to increased airway reactivity [69] . these observations may not be taken as evidence against a role for plasma exudation in asthmatic airways. many characteristics of asthmatic airways can be studied by analyzing the composition and pharmacologic effects of sputum [35] . although their origin was not determined, anaphylatoxins have been identified in asthmatic sputa [98] . asthmatic sputa have been demonstrated to produce smooth muscle contraction [36, 54] and inhibition of ciliary motility [150] . the factors responsible for these effects may well have come from the serum. they may be preformed mediators or mediators produced by activation of plasma proteins at exudation. using chemical analyses menders et al. [83] confirmed the presence of plasma proteins in asthmatic sputa. ryley and brogan [123] showed that the sol phase of asthmatic sputa contained large amounts of albumin and that glucocorticosteroid treatment reduced the plasma protein content along with improvement in lung function [123] . in addition, they studied bronchitic sputa and concluded: this would imply that the sputum of the asthmatic patient had more in common with an inflammatory exudate than that of the chronic bronchitic.., this hypothesis is supported by the finding of a greater proportion of serum albumin and a greater variety of plasma proteins in the asthmatic as compared with the bronchitic sputum [123l. in a more quantitative study brogan et al. [18] confirmed these findings and showed that levels of plasma proteins, but not secretory proteins, were elevated in asthmatic sputa. a high degree of plasma exudate in the airway lumen may also differentiate asthma from emphysema [52] and cystic fibrosis [18] . heilpern and rebuck [55] not only demonstrated high levels of plasma proteins in asthmatic sputa, but also showed that cromoglycate normalized these levels. cromoglycate seems to share with several other antiasthma drugs antileakage effects directly on airway endothelial-epithelial barriers [42, 43] . bronchoalveolar lavage is frequently performed in the clinical evaluation of lung diseases. it is used also in asthma but a relatively large contribution of alveolar liquids to the lavage fluid makes this technique less suitable for the identification of bronchial liquids. this point is illustrated in recent work by lam et al. [75] . they found no difference in albumin levels between normals and asthmatics in large-volume bronchoalveolar lavage liquids. however, using a small volume lavage in a large bronchus they could demonstrate a 10-fold increase of albumin in asthmatic airways compared with controls [75] . although many pieces of information are consistent with the pathophysiological importance of plasma and plasma-derived mediators in airway lumen, this subject has not been extensively reviewed. lord florey, who was experimentally acquainted with the possibility that a considerable amount of fluid may "come directly from the vessels" into the inflamed airway lumen [45] , mentions only in passing, in his excellent chapter on inflammation of mucous membranes, that plasma exudate may seep through the mucosa [44] . in 1882 julius cohnheim [24] discussed how inflammation and plasma exudation might produce different results in different tissues and emphasized that in cavitary organs there may be a passage of exudate across the mucosal barriers. diseases with protein leakage into the gut have received attention due to the ensuing large fall in blood levels of plasma proteins [67] . plasma exudate may escape into the gut lumen through a deranged mucosa and through an apparently intact mucosa [67] . plasma albumin loss due to bronchial diseases has been suggested to occur [15, 53] . for obvious reasons a luminal entry of plasma exudate would have rather more serious consequences for the lower airways than for the gastrointestinal tract. a rapid passage of exudate may increase the depth of the fluid layer in which the cilia beat, and hence cause marked inhibition of mucociliary transport [140] . plasma exudate most likely participates in the formation of mucus plugs: fibrin formation would make the mucus firm and obstructive [34, 61] ; albumin may interact with mucin to form viscous complexes [76] ; plasma proteins may impede normal hydration of mucin [2] . the plasma exudate may enter peripheral airways and compromise the surfactant activity, which in turn may lead to small airway narrowing [77] . it is intriguing that different antiasthma drugs may prevent the mediator-induced microvascular leakage. drug-induced inhibition of vascular leakage can-not be expected to show an immediate reversal of edema because the rate of resolution of interstitial fluid is dependent on lymphatic drainage, which is a relatively slow process. however, if obstructive bronchial tone is dependent on a continuous supply of activated plasma protein mediators from leaky microvessels, an antileakage action might reverse the airway obstruction. it is of interest that protease inhibitors may prevent bronchoconstriction induced by various challenges in asthmatic subjects [105] . in 1940 menkin [84] found that adrenal cortex extract inhibited inflammatory vascular leakage. thirty-three years later leme and wilhelm [74] demonstrated in rats that corticosteron prevents mediator-induced increase in vascular permeability and that this drug inhibits the enhanced venular responsiveness brought about by adrenalectomy. current developments include attempts to identify proteins induced by glucocorticoids. probably by binding with specific receptors followed by induction of anti-inflammatory proteins, glucocorticosteroids inhibit or reduce vascular leakage. in guinea pig airways glucocorticoids such as budesonide may reduce leakage of plasma across both endothelial and epithelial barriers [42] . glucocorticoids have been shown to reduce plasma exudation in inflammatory airway diseases. ryley and brogan [123] found a relationship between a lowering of the albumin concentration in sputa with steroid therapy and clinical improvement in an asthmatic subject. based on serial measurements of neuraminic acid concentration (indicator of bronchial secretion) in asthmatic sputa, keal [68] inferred that the effect of steroid therapy "lies in the reduction of transudate rather than in any change in the bronchial mucosal gland secretion." moretti et al. [92] studied patients with both reversible airways obstruction and bronchitis and showed that 2 weeks' treatment with methylprednisolone brought about a dramatic reduction in the sputum concentration of albumin. stockley and associates [93, 143] examined the effects of about 1 week's therapy with prednisolone on sputum composition in patients with chronic obstructive bronchitis. the patients had no acute chest infections and had, therefore, relatively low levels of serum proteins in their sputa [135] . still, after a few days of treatment a significant reduction was recorded in the ratio of sol-phase sputum concentration to serum concentration of albumin [143] . an antiexudative effect would also reduce the entry of plasma proteins that have protective functions in the airway. the values of c~rantitrypsin followed the same pattern as those of albumin with steroid treatment [143] . however, despite the reduced c~l-antitrypsin levels the inhibitory capacity of the sputum (evaluated on porcine pancreatic elastase) was increased, suggesting that the overall effect of glucocorticosteroids on the airway liquid proteinase-antiprotease balance may be beneficial [93] . findings in nasal washing experiments lend further support to the theory of an antiexudative action of glucocorticosteroids. treatment for 2 days with prednisolone significantly reduced clinical symptoms as well as amounts of albumin (kinins, tame-esterase activity, and histamine) in washings performed during nasal late reaction following challenge with allergen in 13 allergic subjects [118] . this study also produced the interesting information that generation of arachidonate products such as leukotrienes and prostaglandin d was not affected by the steroid treatment [i 18] . attenuation of plasma exudation by glucocorticoids may contribute to the general efficacy of these drugs in asthma and to their potency in inhibiting latephase asthmatic responses and reducing airway hyperresponsiveness. antiasthma xanthines may be subdivided into adenosine-blockers such as theophylline and adenosine-nonblockers such as enprofylline [106, 108] . these xanthines seem to share a number of potentially important pulmonary antiinflammatory effects [see 106] . included among the anti-inflammatory actions is a vascular and epithelial antileakage effect that has been demonstrated in guinea pig airways [42, 106, 112, 113] . it has also been shown that both enprofylline and theophylline prevent the development of pulmonary edema in guinea pigs inhaling histamine [106, 114] . furthermore, antiasthmatic doses of theophylline reduced both symptoms and plasma exudation in human nasal mucosa provoked with different amounts of allergen [96, 108] . cromoglycate also reduced macromolecular leakage across endothelial-epithelial barriers in guinea pigs. this action was not dependent on which mediator had induced the plasma leakage, nor was it due to a reduction of mucosal/submucosal blood flow [42, 107] . the animal data may explain observations in asthmatic subjects reported by heilpern and rebuck [55] 15 years ago. they [55] stated that their study • . . does not attempt to explain why sodium cromoglycate is also effective in non-allergic asthma. however, the evidence points to a previously unrecognized action of the drug, that of lowering albumin concentration in sputum to levels found in non-asthmatic patients. the significance of this finding awaits further study. the possibility that an anti-plasma-leakage action of cromoglycate is important and may compare favorably with other proposed mechanisms of action of the drug in asthma is discussed elsewhere [107] . it has been widely held that the anti-inflammatory effects of sympathomimetic drugs reflect vasoconstriction and diminished blood flow to inflamed tissues. in addition, it has now been demonstrated that these drugs have a vascular antipermeability property. this is fl2-adrenoceptor mediated and more than outweighs the slightly proinflammatory blood flow increasing effect produced by the/3z-receptor agonists [113, 114, 116] . about 10 years ago we demonstrated in guinea pigs that the fl2-receptor agonist terbutaline given systemically or by the inhaled route effectively prevented the development of pulmonary edema induced by subsequent exposure to histamine [114] . this protection might have been via effects on bronchial microvessels, because later studies have demonstrated that terbutaline reduces plasma leakage from the tracheobronchial microcirculation [113] . fl2-receptor mediated antipermeability effects on airway mucosa and microvessels {42, 113] in asthma remain to be examined. a pharmacologic mediator antagonist is by definition effective with some specificity only against 1 type of mediator. thus, antihistamines, leukotriene antagonists, paf-acether antagonists, and tachykinin antagonists among others will specifically antagonize leakage produced by corresponding agonists. since the 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thank mrs lngrid erjeffilt for skillful co-work and my colleagues in lund for encouragement and helpful discussions. i thank mrs ingegerd k~illrn for expert secretarial assistance and mrs marianne persson and her colleagues for excellent library help. key: cord-262776-6k7tcgfs authors: burnouf, thierry; griffiths, elwyn; padilla, ana; seddik, salwa; stephano, marco antonio; gutiérrez, josé-maría title: assessment of the viral safety of antivenoms fractionated from equine plasma date: 2004-09-30 journal: biologicals doi: 10.1016/j.biologicals.2004.07.001 sha: doc_id: 262776 cord_uid: 6k7tcgfs abstract antivenoms are preparations of intact or fragmented (f(ab′)2 or fab) immunoglobulin g (igg) used in human medicine to treat the severe envenomings resulting from the bites and stings of various animals, such as snakes, spiders, scorpions, or marine animals, or from the contact with poisonous plants. they are obtained by fractionating plasma collected from immunized horses or, less frequently, sheep. manufacturing processes usually include pepsin digestion at acid ph, papain digestion, ammonium sulphate precipitation, caprylic acid precipitation, heat coagulation and/or chromatography. most production processes do not have deliberately introduced viral inactivation or removal treatments, but antivenoms have never been found to transmit viruses to humans. nevertheless, the recent examples of zoonotic diseases highlight the need to perform a careful assessment of the viral safety of antivenoms. this paper reviews the characteristics of equine viruses of antivenoms and discusses the potential of some manufacturing steps to avoid risks of viral contamination. analysis of production parameters indicate that acid ph treatments and caprylic acid precipitations, which have been validated for the manufacture of some human igg products, appear to provide the best potential for viral inactivation of antivenoms. as many manufacturers of antivenoms located in developing countries lack the resources to conduct formal viral validation studies, it is hoped that this review will help in the scientific understanding of the viral safety factors of antivenoms, in the controlled implementation of the manufacturing steps with expected impact on viral safety, and in the overall reinforcement of good manufacturing practices of these essential therapeutic products. antivenoms are important biopharmaceutical products made from the plasma of immunized horses or sheep. they are used in human medicine to treat the potentially severe pathophysiological complications resulting from the bites and stings from various animals, such as snakes, scorpions, spiders, cnidarians, lepidopterans or fishes, as well as from intoxications with plants [1, 2] . envenomings are a serious health problem worldwide and are most particularly dreadful in rural areas of the developing world [3] , where shortage of antivenom products [4] and lack of sufficient medical facilities explain numerous fatalities [5, 6] . snake bites represent the major cause of envenoming. case fatality associated with some snake bites reach 50% or more but can be reduced to less than 5% through antivenom therapy, the only available current treatment [5,7e9] . global mortality from snake bites may range from 50,000 to 100,000 per year, but these figures are likely underestimated [5, 10] . viperid snake bite envenoming induces local effects, such as swelling, pain, necrosis, hemorrhage and blistering, often accompanied by secondary infection [9] . systemic viperid envenoming is characterized by a complex pathophysiological profile including coagulopathy, hemorrhage, hypovolaemia, shock and acute renal failure [9, 11] . progressive paralysis may be caused by elapid snake venom neurotoxins, and by some viperid venoms displaying neurotoxic effects. some elapid venoms also induce local necrosis and rhabdomyolysis [9] . snake bite survivors may have major chronic physical and neurological disability. scorpion stings are the second major cause of human fatalities from envenoming (probably amounting to several hundreds per year). scorpion venoms contain toxins which target sodium, potassium, calcium and chloride channels, causing direct effects and release of neutrotransmitters such as acetylcholine and catecholamines, inducing intense local pain and potentially fatal neurotoxic and hemodynamic disturbances [12] . the role of antivenom in the treatment of scorpion stings and other arachnids remains controversial but several reports support their manufacture and use, particularly in severe cases [13, 14] . fatalities have also occurred from envenoming by jellyfish, and venomous fishes. long-term anecdotal experience supports the beneficial effect of stonefish antivenoms [2] , but those may need to be given very early to fight a rapid onset of cardiotoxicity. stings by cnidarians, lepidoptera, centipedes and coneshells and bites by spiders, ticks and one genus of octopus, probably account for about 100 deaths per year [5] . finally, envenomings by massive attacks of africanised bees cause some 30 deaths each year in the americas [5] . antivenoms are most often made by fractionation of plasma of horses (less frequently sheep) that have been immunized with crude venoms [1] . pooled hyperimmune plasma is processed to purify the horse immunoglobulin g (igg) fraction that may be subjected to enzymatic treatment to obtain f(ab#) 2 and fab antibody fragments, and caprylate or ammonium sulphate precipitation to improve their purity [1] . to some extent, some manufacturing steps have features similar to those used to prepare human plasma-derived igg. although there is no known transmission of any infectious agent through antivenoms (albeit under circumstances where rigorous clinical patient follow-up is difficult), theoretical concerns about the possibility of transmission of horse/sheep infectious agents to humans do exist. recent natural transmissions of zoonotic diseases highlight the possible exchanges between humans and the natural reservoirs of biologic agents found in animals [15] , and the inherent risk of emerging diseases [16] . examples of such infections originating from animal (or avian) pathogens include human immunodeficiency virus, ebola, hantaan, lassa, nipah viruses and other paramyxoviruses, equine morbilli virus, west nile virus, and probably severe acute respiratory syndrome (sars) coronavirus [17, 18] . the parenteral transmission of animal viruses to humans is also possible. infectious sv40 virus of rhesus monkeys was a contaminant of early polio vaccines which were administered to a large number of people. whether this was the way in which this virus was introduced into the human population is unclear and a controversial issue. the risk of contamination of antivenoms by equine virus is, therefore, of theoretical concern and has been debated at a recent world health organization (who) workshop [5] . recently, the committee for proprietary medicinal product (cpmp) of the european medicine evaluation agency has published a note for guidance on the manufacture and quality control of animal immunoglobulins and immunsera [19] . however, most manufacturers of antivenoms are located in the developing world and some of them may not be directly exposed to the state-of-the-art process validation concepts, nor have the financial and logistics capability to perform extensive viral validation studies to assess the viral reduction potential of the process they use. therefore, we found it helpful to examine the viral safety of antivenoms by first reviewing the characteristics of equine viruses. we then evaluated the theoretical ability of manufacturing methods of antivenoms to inactivate or remove those viruses, through a comparison with the well validated technologies used to manufacture human plasma-derived igg preparations. finally, we want to emphasize that applying good manufacturing practices in the production of antivenoms, from the control of the animals to that of all production steps, and ensuring traceability in the whole chain of manufacture, represent the current best investment in the quality and safety of these products. horses can harbour enveloped and non-enveloped viruses. the main structural characteristics of those viruses are presented in table 1 ; epidemiological and clinical data and testing methods are described briefly below. eav is a 50e60 nm single-stranded (ss)-rna virus of the arteriviridae family [20] . eav causes an equine viral arteritis, an endotheliotropic viral disease [21] . transmission occurs via respiratory and reproductive routes. there is a variety of clinical signs, and strains vary in virulence [21] , but severe infection can lead to abortions in pregnant mares or neonatal foal death. a one-tube real-time taqman rt-pcr assay was developed for detecting eav. the test was validated using seminal plasma and nasal secretions [22] . a dna vaccine was shown to induce long-term immunization [23] . bdv is a 70e130 nm ss-rna virus belonging to the new bornaviridae family, order mononegavirales. borna disease, known as 'disease of the head', is a sporadically occurring, progressive viral polioencephalomyelitis that primarily affects horses and sheep. after a few weeks to several months incubation, bdv can cause locomotor and sensory dysfunction followed by paralysis and death. bdv exists world-wide in horses, sheep, cattle, cats, dogs and ostriches and can affect a large number of warm-blooded animal species, including humans [24, 25] . the infection can be fatal, but the majority of carriers are asymptomatic [26] . cross-species transmission of this commensal virus has not been proven, but zoonotic aspects of bdv should be considered [26] . bdv-specific antibodies and viral rna have been found in humans with various psychiatric disorders. diagnosis can be made serologically, but also by antigen markers in peripheral white blood cells, combined with nucleic acid amplification [26] . these are closely related 40e70 nm ss-rna alphaviruses [arbovirus type a] of the togaviridae family that are transmitted by arthropods (usually mosquitoes). symptoms of eeev infections, an important multisystemic zoonotic disease, include anorexia and colic, changes in sensorium, hyperexcitability, and terminal severe depression. organ coagulative necrosis and cns lesions are observed [27] . weev is not as neuroinvasive as eeev and the encephalitis caused is not as severe as that caused by eeev. a test to detect eee and wee viral rna has recently been developed [28] . veev is present in both humans and horses but no evidence of transmission from horses to humans by normal routes of contamination has been found. veev has been identified in pharyngeal secretions and is stable when aerosolized; it has been shown to be stable in dried blood and exudates. vaccines have been developed against those three viruses [29] ; one possible case of viral transmission in horse has been suspected following vaccination [30] . ecv is a 75e160 nm ss-rna virus of the coronaviridae family. it has been isolated from feces of a diarrhoeic foal, and has close antigenic and/or genetic relationships with mammalian group 2 coronaviruses [31] . efv, also known as spumavirus, is a 80e100 nm ss-rna virus of the retroviridae family. it belongs to the nonpathogenic, complex unconventional retroviruses and has been isolated in nonhuman primates, cattle, cats, and more recently in the blood of horses [32] . ehvs are large 125e150 nm double-stranded (ds) dna gamma herpes viruses. ehv1 and ehv4 cause much damage to the horse industry and are ubiquitous in the equine population. they are responsible for life-long latent infections in their hosts even in those with natural or vaccine-induced immunity [33] . ehv1 strains are associated with respiratory disease, abortion, and paresis/paralysis, whereas ehv4 strains induce respiratory disease [34] . ehv2 and ehv5 have a less clear pathogenicity and distribution within the equine population. ehv2 may have an aetiological role in ocular disease [35] . the prevalence of ehv2 in adult horses was found to be up to 68% in sweden and 71% in the united kingdom. the prevalence of ehv5 dna was 15 and 24% in adult horses in hungary and the united kingdom, respectively [36] . eiav is a 80e100 nm ss-rna lentivirus of the same retroviridae family as hiv. most infected horses are asymptomatic, with life-long latent infection of leucocytes until stressed (e.g. by pregnancy, corticosteroids, surgical operation, disease) or until new virus variants arise. viraemia then increases by 100-fold. red blood cells become coated by the virus particles and are then lysed by complement, causing jaundice, oedema, hemorrhagic diarrhoea, petechial hemorrhages. eiav can be transmitted from horse to horse by blood. recently a reverse-transcriptase polymerase chain reaction assay (rt-pcr) has been described for quantifying eiav rna in equine plasma [37] . eiv is a 80e120 nm ss-rna virus of the orthomyxoviridae. equine influenza is one of the most economically important contagious respiratory diseases of horses [38] . hev is a 150 nm ss-rna virus [39] . the virus is probably transmitted from bats to horses, and causes natural disease in humans and horses. it is the reference virus for a proposed new genus within the paramyxoviridae family, which also includes another newly identified zoonotic bat virus (nipah), and the salem virus. hev has been recognized in australia as a new zoonotic disease of horses since 1994/1995. this lethal zoonotic viral agent is endemic in certain species of fruit bats (flying-foxes) and over 20% of them in eastern australia are seropositive [40] . six species of flying-foxes in papua new guinea have tested positive for antibodies to hev [40] . hev does not appear to transmit readily between horses under natural and experimental conditions, or from horses to humans [41] . horses can be infected by oronasal routes and can excrete hev in urine and saliva [41] . the virus appears to be spread by close contact with body fluids, such as froth from infected lungs [42] , via nasal discharge, saliva and/or urine [43] . the most important clinical and pathological manifestation of hev infection in horses and humans is severe interstitial pneumonia caused by viral infection of small blood vessels. from 1994 to 1999, epizootic and epidemiological episodes of meningoencephalitis and severe acute respiratory syndromes were reported in australia, malaysia and singapore [15, 44] . it may also cause nervous disease. three incidents of hev disease in horses have been recorded in australiadtwo in 1994, which caused the death of two humans and 15 horses and one in 1999, which involved the death of a single horse [45] . infected horses can develop a severe and often fatal respiratory disease characterized by dyspnoea, vascular endothelial damage and pulmonary oedema. nervous signs may also occur. a number of diagnostic methods have been developed [46] , based on the examination of blood, lung, kidney, spleen, and, if nervous signs are present, also of the brain. pcr assays have been developed. a rapid and sensitive rt-pcr assay using a fluorogenic (taqman) probe was developed to improve the diagnosis of hev infection [47] . both are closely related, arthropod-born, 40e70 nm ss-rna flaviviruses of the flaviviridae family that may affect the central nervous system of horses [48, 49] . evidence of infection with niv was found in the brain of one horse in which inflammation of the meningeal blood vessels occur [42] . niv emerged in malaysia, spread rapidly through the pig population, and caused the deaths of over 100 people [39] . salv has been recently identified in horses [50] . the only known isolate was obtained from a horse that was involved in a disease outbreak of undetermined nature [51] . vsv is a 50e380 nm ss-rna virus of the rhabdoviridae family. it may cause stomatitis in horses, and may represent an emerging equine infectious disease [52] . an immunoglobulin m (igm) capture enzyme-linked immunosorbent assay (mc-elisa) has been developed for the detection of primary infection of vsv in equine and swine sera [53] . wnv is a 40e70 nm ss-rna arbovirus of the flaviviridae family, first identified in the west nile district of uganda in 1937. wnv is found over a broad geographical range and in a wide diversity of vertebrate host and vector species [54] . until 1999, the disease was found in african, european, and eurasian countries. more recently, there was an increase in outbreaks of illness due to wnv in animals as well as humans and numerous cases are currently reported in canada and the usa. infected humans may be asymptomatic and the transfusion of donated blood from such individuals has resulted in the infection and death of recipients. this has led to the introduction in 2003 of strict regulatory measures in canada and the usa which include the testing by a pcr assay of all blood donations. wnv primarily circulates between birds and mosquitoes, while mammalian infections are incidental. mammal biting mosquitoes become infected when they feed on the blood of an infected bird. once this happens, people, horses and other mammals may get wnv. infected horses are, however, unlikely to serve as important amplifying hosts for wnv in nature [55] . many wnv-infected horses probably never show symptoms, but a study reported mortality rates close to 30% [56] . early symptoms are often indistinguishable from other equine encephalitides including rabies, equine herpesvirus-1, equine protozoal myeloencephalitis, and eastern, western, or venezuelan equine encephalomyelitis. a vaccine for horses has been developed, but one horse that received two doses died from the disease [56] . erav and erbv are picornaviruses reclassified as members of the aphtovirus genus because of resemblance to foot-and-mouth disease virus. they are 22e30 nm ss-rna viruses. high neutralizing antibody titers develop and appear to correlate with strong reactivity to vp1 in western blots [57] . a new serotype of the genus erbovirus, tentatively named erbv2 has been identified recently and found in 24% of horses in australia [58] . eev is a 80 nm ds-rna virus of the reoviridae family. several serotypes have been identified in southern africa. serotype-specific virus-neutralizing antibody in serum samples from horses suggests a widespread occurrence of infection but there seems to be only a low level of cross-protection in horses to natural reinfection [59] . erv is a 60e80 nm double-stranded rna of the reoviridae. rotaviruses are important pathogens associated with diarrhoeal diseases in almost all species of mammals. a pcr test with a detection limit of approximately 1.6 ! 10 2 tcid50 per gram faeces, with possible increase in the sensibility by one order of magnitude using nested pcr, was developed, representing a possible diagnostic tool [60] . antivenoms are produced from the plasma or serum collected from immunized horses or sheep. plasma can be obtained by the centrifugation of whole blood or by apheresis. plasma from several animals is typically pooled into 20e300 l batches and subjected to a fractionation process to isolate the igg fraction. following the initial use of crude equine immune serum, still reported to be manufactured by one producer [5] , various methods of igg purification and refinement have been introduced ( table 2 ). most manufacturers use protocols derived from the basic method described by pope [61, 62] , modified by harms [63] , based on pepsin digestion at low ph, to obtain f(ab#) 2 fragments, followed by ammonium sulphate precipitation of antibody fragments [5, 64, 65] . this basic approach is combined with a number of additional steps, aimed at obtaining a purer [66] preparation, such as heat coagulation [64] and ion-exchange chromatography [66, 67] . the ph at which pepsin digestion is performed by various laboratories ranges between 3.1 and 3.8, at temperatures between 30 and 37 c. pepsin digestion is usually performed in undiluted serum or plasma (protein concentration: 60e90 g/l). incubation times range from 40 min to 24 h and with varying pepsin concentrations [64, 65, 67, 68] . thermocoagulation is used by many laboratories and consists of heating at 55e56 c for 1 h [64, 65, 68] although not all methods involving pepsin digestion include heat coagulation [67] . some fractionation protocols include an additional table 2 steps used for the manufacture of antivenoms screening of production animals for adventitious agents plasma collection (whole blood or apheresis) in bags or bottles plasma thawing at room temperature plasma pooling igg/fragments purification process f(ab#) 2 : b pepsin digestion (acid ph) and ammonium sulphate precipitation, or b pepsin digestion (acid ph) and caprylic acid precipitation fab: ammonium sulphate precipitation and papain digestion at ph 7e8 whole igg: b caprylic acid precipitation b ammonium sulphate precipitation igg/fragments concentration: (nh 4 ) 2 so 4 /na 2 so 4 precipitation, ultrafiltration polishing: ion exchange (removes fc and further purifies iggs/ fragments) ultrafiltration sterile filtration aseptic filling storage in the liquid state or lyophilisation acidification step to remove some non-igg globulins ('euglobulins') which are unstable at acid phs [66, 69] . one producer includes a bulk pasteurisation step in its antivenom production protocol [66] . other manufacturers produce whole igg antivenoms using either ammonium sulphate precipitation of igg or caprylic acid precipitation of non-igg proteins [69e71], followed by dialysis or ultrafiltration. recently, a simple, one-step methodology based on caprylic acid precipitation has been described [72] . the conditions used for caprylic acid fractionation of equine plasma are as follows: plasma ph is adjusted to 5.8 and caprylic acid is added directly to this plasma to attain a final concentration of 5% (v/v). the mixture is stirred during caprylic acid addition and then for one additional hour, after which the precipitate is separated by filtration. caprylic acid is then removed by either dialysis or ultrafiltration and the product is formulated before sterile filtration [72] . some antivenoms are made of fab antibody fragments, obtained by papain digestion of sheep igg, at neutral ph, after sodium sulphate precipitation of igg [73] . affinity-chromatography or ion-exchange chromatography steps have been described in the production of some fab antivenoms [74, 75] . treatment with b-propiolactone has been evaluated to reduce complement activation by horse plasma-derived products [76] ; however, it does not appear to be used routinely in laboratories producing antivenoms. in addition to the differences in fractionation protocols, antivenoms made of whole igg, f(ab#) 2 and fab molecules greatly differ in their pharmacokinetic profiles [1, 77] . most antivenoms come in liquid presentation, but some are lyophilized. the former need to be stored at 4e8 c, since higher temperatures reduce their shelf-life and induce protein denaturation and aggregate formation [78] . in contrast, lyophilized antivenoms can be stored at room temperature and have a more prolonged shelf-life. most antivenoms contain preservatives to prevent bacterial contamination [5, 79] . considerable experience has been gained in recent years on the viral safety of human igg preparations and other human plasma-derived products. historical perspectives on these products help in assessing the viral safety of antivenoms. the safety of human plasma products results from the combination of overlapping strategies that include: (a) selection of donors, (b) viral testing procedures on single donations (e.g. serological tests) and plasma pools (e.g. nucleic acid tests) to exclude donations contaminated by viruses, (c) processing and purification steps to inactivate and remove viruses, and (d) implementation of good manufacturing practices [80] . whenever feasible, similar approaches should be used for the manufacture of animal plasmaderived products. monitoring the health of animals used for antivenom production is important [5] . colonies of animals should be kept free of contamination through good husbandry or vaccination, where appropriate. surveillance of the source animals should include screening for pathogens, health monitoring, including routine blood chemistry and haematology tests, and post-mortem examination. ideally, animals should be kept under strictly contained conditions, but this is not readily applicable to horses. however, for instance, animals could be kept free of a particular arthropod-borne virus if they are maintained in an area free of the particular arthropod vector. if a particular pathogen is identified, it should be established whether it is present in plasma. some virological screening methods may be performed, if available and epidemiologically relevant to a particular geographic region, to limit risk of virus presence in the animal herd and/or the plasma pool. to some extent the guidelines from the federation of european laboratory animal science association (felasa), although not referring to horses, may serve as a reference [81e83]. nevertheless, not all of these measures are available in many developing countries nor readily applicable to horses used for antivenom production, and pathogens can escape this surveillance program. this illustrates the need for manufacturing techniques to ensure a sufficient margin of safety with regards to potential infectious agents present in the manufacturing plasma pool. this also strongly emphasizes the crucial importance of ensuring an appropriate traceability system from horse donors to human recipients of antivenoms so that any potential infectious events or risks may be identified in a proper and timely manner and relevant counter-measures taken promptly. confidence in the safety of antivenoms must come from evidence that the manufacturing processes used can reproducibly remove or inactivate high potential levels of viruses. comparison with human plasma fractionation suggests that some of the steps of antivenoms include treatments which may inactivate viruses. human plasma fractionation is a complex integrated manufacturing process from which products like albumin, coagulation factors and igg, are obtained. purification processes combine cryoprecipitation, ethanol fractionation, chromatography, and ultrafiltration [84] . in addition, various types of dedicated viral reduction treatments have been introduced, most often on purpose, such as chemical treatments by solvent-detergent, caprylic acid precipitation, low-ph incubation, heat treatments in the liquid or in the dry states, and nanofiltration [80] . among those, caprylic acid and low ph treatments, both of which are commonly used also for the purification of antivenom igg, have been shown to contribute to the viral safety of human plasma igg products as described below. some years ago, unsaturated fatty acids were shown experimentally to inactivate more than 4 log of lipidenveloped viruses (vsv, sindbis virus, hiv) in human plasma protein fractions [85] . however, specific attention has been given to caprylic acid (also called octanoic acid), as it has been used as precipitating agent for human igg. it has been suggested that the non-ionized form of the caprylic acid disrupts the integrity of the lipid bilayer and membrane associated proteins of enveloped viruses. utilizing the dissociation reaction and varying the concentration of the ionized form of caprylate, a specific amount of the nonionized form of caprylate can be maintained over a wide ph range. treatment of plasma protein solution with caprylate at acidic ph also readily inactivates lipid-enveloped viruses like herpes simplex virus type i, vesicular stomatitis virus, vaccinia virus, and sindbis virus [86] . detailed validation studies of two caprylic acid treatments of human ig have been published recently. the robustness of a treatment applied to three intravenous immunoglobulin preparations (igg, igm-enriched, and igm preparations) has been investigated using hiv, bvdv, sindbis virus and pseudorabies as model viruses [87] . the routine treatment conditions for these two igg and this igm preparation are indicated in table 3 . kinetics of inactivation was determined over a period of 1 h of treatment. complete inactivation (corresponding to more than 4.68e6.25 log) is achieved within the first minutes. within a certain range, viral inactivation in the igg product was not affected by ph (5.5e6.0), temperature (0e26 c), and protein content (30e40 g/l). above ph 6, and most specifically at ph 8, no bvdv inactivation was found. at a content of caprylic acid of 3.7 g/kg or less, inactivation of hiv is significantly reduced. under the conditions applied during manufacture, caprylic acid leads to robust inactivation of lipid-enveloped viruses; ph is a particularly critical parameter and should be less than 6. these conditions have also been found to inactivate o4.7 log10 eav, an equine virus used as a model (dichtelmu¨ller, personal communication). another study reported the viral inactivation achieved during caprylic acid precipitation of non-igg proteins from human igg product [88] . at ph 5.1, 23 c, and in the presence of 9 mm caprylate, r 4.7 and r 4.2 log of hiv and prv, respectively, were inactivated during the 1 h treatment, but only 1.5 log for bvdv was inactivated. at 12 mm caprylate, r 4.4 log of bvdv was inactivated within this time period. at ph 5.1, 24 c, 19 mm caprylate, and ph 5.1, 24 c, 12 mm caprylate, complete inactivation of bvdv and of hiv and prv was achieved in less than 3 min. the authors also showed that 40 mm caprylate at ph 5.4 and 40 c in supernatant iv-1, an intermediate in albumin production, inactivated r 4.2 log bvdv almost instantaneously [88] . the virucidal effect of caprylic acid has also been confirmed in human albumin solution, where it is used as a stabilizer for pasteurisation. elevated temperature and low ph were found to be critical parameters to ensuring significant reduction in virus infectivity. rate and extent of inactivation were sensitive to variations in the caprylate to protein ratio and to changes in ph. in the conditions retained, 10% w/v protein, 16 mm caprylate, ph 4.5 and 30 c, more than 4 log inactivation of the lipid-enveloped viruses tested, including bvdv, was achieved [89] . it should be kept in mind that treatment of whole plasma or crude fractions, as is the case for equine antivenoms production, may lead to lower rate and kinetics of viral inactivation, due to the high endogenous lipid content, as found in a study that evaluated the virucidal effect of sodium oleate [85] . finally, one should keep in mind that caprylic acid treatment does not inactivate non-enveloped viruses. several human igg preparations are subjected to an acid ph incubation that was historically introduced to reduce immediate adverse reactions subsequent to intravenous injections. in the late 1980s, acid ph treatment was also shown to contribute greatly to the table 3 comparison of conditions for caprylic acid treatment used for human igg preparations (87) [90, 91] . the treatment of human igg generally consists of incubation at ph 4, with or without traces of pepsin, at temperatures from 30 to 37 c, using a protein content close to 50 g/l, and for more than 20 h. the treatment is intended to eliminate aggregates. pepsin, when present, is maintained at low concentration to avoid cleavage of the igg molecule. several model enveloped viruses were shown to be inactivated under those conditions [92e94]. more than 5e8 log inactivation of hiv and other enveloped viruses used as surrogate models, such as semliki forest virus (sfv), hsv, vsv, and cmv is achieved. by contrast, poliovirus, as other non-enveloped virus, seems more resistant. the presence of trace concentrations of pepsin added to reduce anticomplementary activity during this procedure has been shown to contribute little to virus kill of most processes [95] . virus inactivation by acid ph and pepsin is influenced by several parameters. hiv, bvdv, sfv, and prv are completely inactivated within 5 mine1 h at 37 c but inactivation rate and extent is less at lower temperatures. increasing the sucrose content from 0 to 15% reduced the rate of inactivation of prv but not that of sfv. increasing the nacl content from 0 to 150 mm reduced the rate of inactivation of sfv but that of prv was unchanged. an increase in the igg concentration from 0 to 10% speeds up the inactivation of prv but decreases that of sfv. therefore, temperature is a major parameter in the virucidal efficacy of ph-pepsin treatment and the impact of solute composition is virus-dependent [93] . some fractionation protocols used in antivenom production include a low ph treatment to induce the precipitation of 'euglobulins', which are plasma globulins unstable under these conditions. this step was shown to remove between 2.8 and 3.5 log of eiav, sindbis virus, poliovirus and porcine parvovirus [66] . virus reduction may occur by co-precipitation with the discarded 'euglobulins' rather than by an inactivation process [66] . pasteurisation is the treatment of a liquid protein fraction for 10 h at 60 c [96] and is being used in the production of at least one type of equine-derived antivenom [66] . experience with human plasma products show that liquid heat treatment may inactivate both enveloped and non-enveloped viruses. pasteurisation of albumin solutions is carried out in the final container in the presence of low concentrations of sodium caprylate alone or with n-acetyl tryptophan. inactivation of sindbis and emc model viruses added to 5% albumin solution was achieved within 10 min treatment [97] . however, inactivation of some non-enveloped viruses is less [80] , and the process appears to be virus-specific. various coagulation factors, protease inhibitors, and intravenous igg are pasteurised during the purification process. most often, pasteurisation is performed in the presence of stabilizers, like amino acids, sugars, or citrate [97, 98] , to protect protein functionality, and limit molecular alterations and protein aggregation. protein stabilizers are also known to stabilize viruses, however, further highlighting the need for validating the exact conditions of treatment used. pasteurisation can inactivate viruses of different types, enveloped or nonenveloped, including hiv, hbv, hcv, and hav [80, 96, 98, 99] . there are few published data on the inactivation of resistant model non-enveloped viruses like porcine parvovirus, sv 40 or reovirus type 3 in plasma products [100, 101] . in most instances, inactivation of such viruses is limited to less than 2e4 log over the 10 h heating period. heat treatment of whole human plasma at 50 c for 3 h, in the presence of a specific combination of stabilizers, inactivates more than 6 log of hiv, and more than 4 log of enveloped and nonenveloped model viruses [102] . finally, it was demonstrated that heat treatments like pasteurisation and vapour heat treatment (as well as solvent-detergent and nanofiltration on 35 nm membranes) can inactivate/ remove more than 6 log of wnv [103] . chromatography is primarily used in human plasma fractionation as a downstream polishing step [104] . ionexchange chromatography has been described for some antivenom products [66, 67] . viral partitioning has been shown to occur during affinity and ion-exchange chromatography, but the mechanism of removal is not easy to predict and control [105] , making it difficult to consider these as robust viral removal steps. storage at ph 4.25, in the presence of a stabilizer, such as 10% maltose, for a minimum of 21 days at 20 c, yields aggregate-free and in vitro functionally active human igg preparations [106] . incubating at ph 4.25 for 21 days at 20 c caused a 10,000-fold decrease in bvdv infectivity and complete inactivation of 1000 chimpanzee infectious doses per millilitre of hcv [107] . with the exception of a recent report of a fab ovine antivenom formulated at ph 4.0 [108] , to our knowledge, formulation at low ph is not commonly used for antivenoms but may be considered as a viral safety step, if it does not affect the biological activity, the general safety, and the clinical efficacy of the product. viral validation studies are small-scale experiments designed to provide an estimate of the overall viral reduction level achieved across the manufacturing processes and to identify steps and parameters that are critical for viral inactivation or removal. to perform such studies, as described in the guidelines prepared by the cpmp, production intermediates are voluntarily spiked with known amounts of known viruses, using laboratory-scale mimicking of the production process, and the virus reduction factor is calculated by comparing the infectivity level before and after each individual process step evaluated. when appropriate, a cumulative viral reduction factor can be calculated and provides information on the viral safety margin of processes for a range of model viruses. effective and robust individual steps typically achieve more than 4 log reduction in infectivity under a large range of production parameters. viral validation studies are usually conducted with 3e6 model viruses differing in their structural features (presence of an envelope, rna or dna genetic material, size, degree of resistance). ''relevant'' viruses, which are known potential contaminant of the starting plasma, should be used when possible. a list of existing laboratory adapted model viruses that could be used for validation studies of antivenoms manufacturing processes is shown in table 4 . vsv and wnv that may contaminate horses appear of special relevance for process validation of equine products. dedicated viral inactivation and removal production steps should be implemented in a way to ensure the reproducibility in viral reduction and the absence of risks of downstream contamination. examples of approaches used for the manufacture of human plasma products are provided in recent who guidelines [109] , and a cpmp note for guidance provides recommendation on production of immunsera [19] . both can be used as reference by manufacturers of antivenoms. viral reduction equipment should have adapted specifications. for instance, equipment for bulk in process virus inactivation, such as the acid ph incubation or the caprylic acid treatment, should ideally be fully enclosed. in addition, vessels should have temperature controls and be designed to avoid ''dead points'' where important parameters such as temperature and ph could not be ensured. similarly, liquid heat treatment or heat shock should be conducted in jacketed tanks with the solution stirred throughout the heat cycle to ensure homogeneity and all points in the tank should be within the specified temperature range. during the qualification phases, the equipment and the required services should be shown to conform to the predefined technical specifications and requirements, and to function within the specified limits. when possible, it is recommended to create a dedicated ''viral safety area'' where production steps are arranged in a clear and logical way, so that recontamination of the virally reduced intermediates is avoided. operating procedures should also be written to reduce the likelihood of crosscontamination. bulk ph 4 or caprylic acid virus inactivation could be carried out in two stages, the first stage being located in a normal production room, followed by a second incubation in another tank located in a segregated, contained area. the duration of the first stage should be such that the majority of virus inactivation (as found during the viral validation studies) has occurred. on completion of the first stage, the product should be aseptically transferred (e.g. through sterile coupling) into the second vessel located in a safety zone for completion of the second stage of viral inactivation. product-contacting-equipment used in the safety area should be dedicated or decontaminated to inactivate any remaining virus. a quality assurance system should ensure that the execution of virus reduction methods conforms to the validated conditions. viral inactivation and removal procedures should be described in approved standard operating procedures (sops) that contain critical process limits for the viral inactivation and removal methods applied to antivenoms. to date there is no documented example of viral transmission to humans from the use of antivenoms, whichever type of product (whole igg, f(ab#) 2 or fab) is manufactured [5] . considering the difficulty in the containment of large animals like horses, and in an exhaustive viral testing of the starting plasma, the viral safety of antivenoms appears therefore largely dependent upon the capacity of manufacturing processes to reduce infectious agents and on the use of good manufacturing practices, in particular in ensuring production traceability. by contrast to human plasma products, most antivenoms are currently not subjected to purposely introduced viral reduction steps like solvent-detergent, pasteurisation, or nanofiltration [80, 109, 110] . however, a comparison with validated manufacturing processes used for human igg clearly indicates that at least two widely used antivenom production steps, caprylic acid treatment and low-ph incubation, are likely to contribute in a robust manner to viral safety, at least against enveloped viruses. the concentration of caprylic acid (5%) used for antivenoms is higher than that used for human igg (table 3) , and the ph of treatment is within the effective range (less than ph 6) required for optimal inactivation of enveloped viruses. as the treatment is performed on equine plasma itself, or on crude fractions, the viral kill could be reduced by the presence of lipids. the acid incubation of antivenoms which is performed at a ph (3.1e3.8), less than that used for human igg, and at a similarly warm temperature (30e37 c) ( table 5) should provide a fast rate of inactivation of lipidenveloped viruses, and possibly also non-enveloped viruses. however, the incubation time (40 mine24 h) is for some processes significantly less than for human igg (typically 22e25 h), probably reducing the extent of viral kill achieved. preliminary viral kill data obtained with antivenoms confirm that conditions used for peptic cleavage at low ph of f(ab#) 2 achieve robust inactivation of wnv [111] . we recommend that further representative viral validation studies using a range of well-established virus models are performed to know the rate and extent of viral kill actually achieved during those production steps. the viruses to use for those validations should be discussed with specialized virologists, and, by analogy with most studies done with human plasma derivatives, might include three lipidenveloped viruses (such as bvdv, vsv, psr, and wnv) and one non-enveloped virus (such as emc, poliovirus, or reovirus 3). inactivation data could then be compared to viral kills achieved using similar process steps of human igg products, therefore providing some reference on their robustness and allowing manufacturers and national regulatory authorities to assess scientifically the margin of safety of antivenom products. other manufacturing steps, such as heat coagulation, could be validated as well. formulation of the product at low ph, as done for some human igg preparations, is another potential approach to consider for improved viral safety, but evaluating product stability and efficacy under such conditions would be a pre-requisite. in view of their expected beneficial impact on viral safety, appropriate process implementation of caprylic acid treatments and low-ph incubations should be ensured, as well as measures taken to avoid risks of downstream contamination and, more widely, to respect good manufacturing practices and traceability concepts. such conclusions should also be valid for other animal plasma-derived igg products used in human medicine such as anti-lymphocyte/t-cell, antitoxins, and anti-bacterial or viral agents sera [19] . snake antivenoms marine antivenoms venomous bites and stings a new pan african polyspecific antivenom developed in response to the antivenom crisis in africa report of a who workshop on the standardization and control of antivenoms venoms, antivenoms and immunotherapy efficacy, safety, and use of snake antivenoms in the united states antivenoms: a list of hyperimmune sera currently available for the treatment of envenoming by bites and stings handbook of clinical toxicology of animal venoms and poisons snake-bites: appraisal of the global situation clinical toxicology of snakebite in central america handbook of clinical toxicology of animal venoms and poisons development of an improved method for production of antiscorpion f(ab#)2 fragment of igg with high yield and potency antivenom treatment in arachnidism emergence of transmissible disorders, a continuous processda new type of viral meningoencephalitis recent developments in the epidemiology of virus diseases possible role of an animal vector in the sars outbreak at amoy gardens biologists seek to head off future sources of infection note for guidance on production and quality control of animal immunoglobulins and immunsera for human use. london: european agency for the evaluation of medicinal products (emea) structure of arterivirus nsp4. the smallest chymotrypsin-like proteinase with an alpha/beta c-terminal extension and alternate conformations of the oxyanion hole growth characteristics of a highly virulent, a moderately virulent, and an avirulent strain of equine arteritis virus in primary equine endothelial cells are predictive of their virulence to horses detection of equine arteritis virus by real-time taqman reverse transcription-pcr assay stable and long-lasting immune response in horses after dna vaccination against equine arteritis virus borna disease: current knowledge and virus detection in france borna disease virus and infection in humans borna disease virus: new aspects on infection, disease, diagnosis and epidemiology clinical, pathologic, immunohistochemical, and virologic findings of eastern equine encephalomyelitis in two horses detection of north american eastern and western equine encephalitis viruses by nucleic acid amplification assays immune response of horses after simultaneous or sequential vaccination against eastern, western, and venezuelan equine encephalomyelitis eastern equine encephalomyelitis virus infection in a horse from california characterization of a coronavirus isolated from a diarrheic foal further characterization of equine foamy virus reveals unusual features among the foamy viruses detection and isolation of equine herpesviruses 1 and 4 from horses in normandy: an autopsy study of tissue distribution in relation to vaccination status equine herpesvirus 1 and 4 infections: an update detection of equine herpesvirus type 2 (ehv-2) in horses with keratoconjunctivitis prevalence of equine herpesvirus types 2 and 5 in horse populations by using type-specific pcr assays development of a multiplex real-time reverse transcriptase-polymerase chain reaction for equine infectious anemia virus (eiav) equine influenza virus infections: an update the exceptionally large genome of hendra virus: support for creation of a new genus within the family paramyxoviridae newly discovered viruses of flying foxes transmission studies of hendra virus (equine morbillivirus) in fruit bats, horses and cats hendra and nipah virus infections the australian paralysis tick may be the missing link in the transmission of hendra virus from bats to horses to humans infection of humans and horses by a newly described morbillivirus hendra virus disease in horses hendra (equine morbillivirus) development of a fluorogenic rt-pcr assay (taqman) for the detection of hendra virus japanese encephalitis in horses in japan japanese b encephalitis in a horse identification and phylogenetic comparison of salem virus, a novel paramyxovirus of horses isolation of salem virus, a novel equine paramyxovirus, and assessment of its etiologic role in a disease outbreak vesicular stomatitis and other vesicular, erosive, and ulcerative diseases of horses development of an immunoglobulin m (igm) capture enzyme-linked immunosorbent assay for detection of equine and swine igm antibodies to vesicular stomatitis virus west nile virus in livestock and wildlife experimental infection of horses with west nile virus west nile virus encephalomyelitis in horses: 46 cases equine rhinitis a virus: structural proteins and immune response equine rhinitis b virus: a new serotype the classification of seven serotypes of equine encephalosis virus and the prevalence of homologous antibody in horses in south africa nested reverse transcriptase-polymerase chain reaction for the detection of group a rotaviruses the action of proteolytic enzymes on the antitoxins and proteins in immune sera. i. true digestion of the proteins the action of proteolytic enzymes on the antitoxins and proteins in immune sera. ii. heat denaturation after partial enzyme action the purification of antitoxic plasmas by enzyme treatment and heat denaturation antivenins in brazil: preparation antivenin production in india preparation of improved f(ab#)2 antivenoms. an example: new polyvalent european viper antivenom (equine) a protocol for 'enhanced pepsin digestion': a step by step method for obtaining pure antibody fragments in high yield from serum quantitative comparison on the refinement of horse antivenom by salt fractionation and ionexchange chromatography manual de procedimientos. produc-cio´n y pruebas de control en la preparacio´n de antisueros difte´rico purification of f(ab#)2 anti-snake venom by caprylic acid: a fast method for obtaining igg fragments with high neutralization activity, purity and yield a randomized blinded clinical trial of two antivenoms, prepared by caprylic acid or ammonium sulphate fractionation of igg, in bothrops and porthidium snake bites in colombia: correlation between safety and biochemical characteristics of antivenoms caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production a new antivenom to treat eastern coral snake (micrurus fulvius fulvius) envenoming first clinical experiences with specific sheep fab fragments in snake bite first clinical experiences with a new ovine fab echis ocellatus snake bite antivenom in nigeria: randomized comparative trial with institute pasteur serum (ipser) africa antivenom effect of beta-propiolactone treatment on the complement activation mediated by equine antisera pharmacokinetic-pharmacodynamic relationships of immunoglobulin therapy for envenomation effect of storage temperature on the stability of the liquid polyvalent antivenom produced in costa rica effect of preservatives on igg aggregation, complementactivating effect and hypotensive activity of horse polyvalent antivenom used in snakebite envenomation reducing the risk of infection from plasma products: specific preventative strategies recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units felasa recommendations for the health monitoring of experimental units of calves, sheep and goats report of the federation of european laboratory animal science associations (felasa) working group on animal health report of the federation of european laboratory animal science associations (felasa) working group on animal health accepted by the felasa board of management chromatography in plasma fractionation: benefits and future trends inactivation of lipid-enveloped viruses in labile blood derivatives by unsaturated fatty acids inactivation of lipid-enveloped viruses in proteins by caprylate inactivation of lipid enveloped viruses by octanoic acid treatment of immunoglobulin solution enveloped virus inactivation by caprylate: a robust alternative to solvent-detergent treatment in plasma derived intermediates low ph, caprylate incubation as a second viral inactivation step in the manufacture of albumin. parametric and validation studies potential contribution of mild pepsin treatment at ph4 to the viral safety of human immunoglobulin products the viral safety of intravenous immune globulin virus validation of ph 4-treated human immunoglobulin products produced by the cohn fractionation process virus inactivation by pepsin treatment at ph 4 of igg solutions: factors affecting the rate of virus inactivation virus inactivation during intravenous immunoglobulin production viral safety of intravenous immunoglobulins g for therapeutic use strategies to produce virus-safe blood derivatives inactivation of viruses in labile blood derivatives. ii. physical methods pasteurization as an efficient method to inactivate blood borne viruses in factor viii concentrates inactivation of hiv, hbv, hcv related viruses and other viruses in human plasma derivatives by pasteurisation a pasteurized therapeutic plasma pasteurization of antihemophilic factor and model virus inactivation studies virucidal heattreatment of single plasma units: a potential approach for developing countries nile virus and the safety of plasma derivatives: verification of high safety margins, and the validity of predictions based on model virus data affinity chromatography in the industrial purification of plasma proteins for therapeutic use chromatographic removal of viruses from plasma derivatives a comparative in vitro study of low ph and enzyme treated immunoglobulin preparation for intravenous use inactivation of hepatitis c virus in low ph intravenous immunoglobulin formulation of a liquid ovine fab-based antivenom for the treatment of envenomation by the nigerian carpet viper (echis ocellatus) world health organization expert committee of biological standardization. guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products. www.who.int/biologicals-quality%20assur-ance%20and%20safety nanofiltration of plasma-derived biopharmaceutical products inactivation of west-nile virus during peptic cleavage of horse plasma igg we express our sincere thanks to dr. herbert dichtelmu¨ller for his review of the manuscript and expert suggestions. key: cord-266147-s8rxzm0t authors: burnouf, thierry title: modern plasma fractionation date: 2007-03-28 journal: transfus med rev doi: 10.1016/j.tmrv.2006.11.001 sha: doc_id: 266147 cord_uid: s8rxzm0t protein products fractionated from human plasma are an essential class of therapeutics used, often as the only available option, in the prevention, management, and treatment of life-threatening conditions resulting from trauma, congenital deficiencies, immunologic disorders, or infections. modern plasma product production technology remains largely based on the ethanol fractionation process, but much has evolved in the last few years to improve product purity, to enhance the recovery of immunoglobulin g, and to isolate new plasma proteins, such as α1-protease inhibitor, von willebrand factor, and protein c. because of the human origin of the starting material and the pooling of 10 000 to 50 000 donations required for industrial processing, the major risk associated to plasma products is the transmission of blood-borne infectious agents. a complete set of measures—and, most particularly, the use of dedicated viral inactivation and removal treatments—has been implemented throughout the production chain of fractionated plasma products over the last 20 years to ensure optimal safety, in particular, and not exclusively, against hiv, hepatitis b virus, and hepatitis c virus. in this review, we summarize the practices of the modern plasma fractionation industry from the collection of the raw plasma material to the industrial manufacture of fractionated products. we describe the quality requirements of plasma for fractionation and the various treatments applied for the inactivation and removal of blood-borne infectious agents and provide examples of methods used for the purification of the various classes of plasma protein therapies. we also highlight aspects of the good manufacturing practices and the regulatory environment that govern the whole chain of production. in a regulated and professional environment, fractionated plasma products manufactured by modern processes are certainly among the lowest-risk therapeutic biological products in use today. protein products fractionated from human plasma are an essential class of therapeutics used, often as the only available option, in the prevention, management, and treatment of life-threatening conditions resulting from trauma, congenital deficiencies, immunologic disorders, or infections. modern plasma product production technology remains largely based on the ethanol fractionation process, but much has evolved in the last few years to improve product purity, to enhance the recovery of immunoglobulin g, and to isolate new plasma proteins, such as a1protease inhibitor, von willebrand factor, and protein c. because of the human origin of the starting material and the pooling of 10 000 to 50 000 donations required for industrial processing, the major risk associated to plasma products is the transmission of blood-borne infectious agents. a complete set of measures-and, most particularly, the use of dedicated viral inactivation and removal treatments-has been implemented throughout the production chain of fractionated plasma products over the last 20 years to ensure optimal safety, in particular, and not exclusively, against hiv, hepatitis b virus, and hepatitis c virus. in this review, we summarize the practices of the modern plasma fractionation industry from the collection of the raw plasma material to the industrial manufacture of fractionated products. we describe the quality requirements of plasma for fractionation and the various treatments applied for the inactivation and removal of blood-borne infectious agents and provide examples of methods used for the purification of the various classes of plasma protein therapies. we also highlight aspects of the good manufacturing practices and the regulatory environment that govern the whole chain of production. in a regulated and professional environment, fractionated plasma products manufactured by modern processes are certainly among the lowest-risk therapeutic biological products in use today. a 2007 elsevier inc. all rights reserved. c ollected human plasma may be used as a therapeutic product (known as bclinical plasmaq or bfresh frozen plasmaq) or as source material for the production of pharmaceutical fractionated products (also called bplasma productsq or bplasma derivativesq). this complex biologic material contains hundreds of proteins covering a myriad of physiological functions. many components still have undiscovered roles. the most abundant proteins, albumin and immunoglobulin (ig) g, are present at about 35 and 10 g/l, respectively, representing about 80% of all plasma proteins. less abundant proteins include the protease inhibitors, like a1-antitrypsin (aat) (1.5 g/l) and antithrombin (at) (300 mg/l), and the coagulation factors such as factor viii (fviii) (a few ng/l), which exhibit potent physiologic activity. currently, about 20 different plasma protein therapeutics are used for treating life-threatening diseases or injuries associated to bleeding and thrombotic disorders, immunological diseases, infectious conditions, as well as tissue degenerating diseases, thus addressing the clinical needs of countless patients. an updated list of the major therapeutic applications of plasma protein products can be found elsewhere. 1 this industrial process used to isolate therapeutic plasma proteins is known as bfractionation.q over 23 to 28 million liters of human plasma are fractionated each year in the world, in batches of several thousand liters, in about 70 factories. modern plasma fractionation combines manufacturing steps to isolate, in a sequential and integrated manner, the crude fractions that are further purified into individual therapeutic products. validated dedicated steps inactivate and/or remove infectious agents potentially present in the starting plasma pool. this sophisticated industrial process is performed under highly hygienic conditions in licensed facilities (plasma fractionation plants) that are operated in compliance with good manufacturing practices and following quality assurance principles. over the years, plasma fractionation has evolved from a medical service activity mostly oriented toward the needs of local communities into a global manufacturing industry conforming to high regulatory standards. these strict requirements start from the collection of plasma for fractionation and include product manufacture and distribution steps. in this article, we review the most current practices encompassing the collection of plasma for fractionation, the core industrial plasma fractionation process, and the purification and pathogen reduction technologies of individual plasma products. the practices used for the collection of plasma for fractionation have direct influence on the safety profile of protein products since individual donations contribute to large plasma pools used to manufacture therapeutic preparations intended for hundreds or even thousands of patients. it is therefore logical that the production of plasma is regarded as an integral part of the manufacture of modern fractionated products. collection requirements of plasma for fractionation may differ from those relevant to fresh frozen plasma. in a regulated environment, plasma for fractionation is collected by licensed/registered blood establishments (blood centers and apheresis collection centers) that are inspected by the relevant national regulatory authorities (nras). compelled by the same safety concerns, the plasma fractionators conduct audits to verify that the contractual plasma collection and quality and safety measures, agreed upon with the plasma supplier, are met. areas of specific relevance include (a) procedures for donor screening and donation testing; (b) labeling, documentation, and traceability requirements; and (c) the handling of blood and plasma. such information is part of the marketing license of plasma products and, in europe, is assembled into a document called the bplasma master file.q various requirements for the collection of plasma for fractionation have been described in various guides, eg, from the pharmaceutical inspection convention and pharmaceutical inspection cooperation scheme (jointly referred to as pic/s), us food and drug administration (fda) and in recent world health organization recommendations. 1 they are summarized below. candidate donors are provided with educational materials and undergo a medical interview to establish the absence of risks or signs of infections and to prove compliance for a donation of plasma for fractionation (table 1) . potential donors presenting a health hazard are asked to exclude themselves. medical information of donors is acquired and archived. continuous epidemiologic surveillance of the donor population is being required in some jurisdictions. 2 it helps to establish the background level (prevalence and incidence) and trends of known infectious markers (eg, hiv 1 and 2 antibodies, hepatitis c virus [hcv] antibodies, and hepatitis b surface antigen [hbsag]) in the population. this is also of interest for the early detection of emerging diseases, allowing early implementation of counter measures (such as more stringent donor screening processes or requirements for additional testing procedures). donors eligible to donate plasma for fractionation are individuals who meet donation criteria (such as age and donation frequency), do not present risk factors of blood-born infectious agents, and comply with requirements defined by the plasma fractionator and the nras of the country of plasma collection and of use of the products. in most situations, eligibility of whole blood donors and apheresis donors overlap, apart from donation frequency which is higher for plasmapheresis donors. eligibility criteria take into account scientific information about the risks of transmission of infectious agents by pooled plasma products (which may differ from those by blood components). special criteria may exist for the collection of hyperimmune plasma (used to make hyperimmune igg preparations), such as procedures for donors' immunization and minimal antibody titer. 1 currently, about 35% of the plasma fractionated in the world is obtained by centrifugation of whole blood (brecoveredq plasma), and 65% is obtained by apheresis. blood/plasma collection, processing, and storage may affect plasma quality, as well as having an impact on the recovery of the most labile proteins such as fviii. in particular, risks of activation of the coagulation, complement, and fibrinolytic systems, which may lead to generation of plasma proteases, should be avoided. to better preserve the integrity of recovered plasma and limit risks of activation of the coagulation cascade and of cellular components, (a) good mixing of the blood with the anticoagulant solution (a sodium citrate based solution 1 ) should be ensured from the initiation till the end of the collection process; (b) the duration of the collection should not exceed 15 minutes; and (c) temperature variations of the blood should be avoided. a few hours after donation, whole blood is subjected to a centrifugation that separates the cellular elements (most specifically red cells) from plasma. the mean plasma volume obtained from one whole blood donation is about 220 ml but varies depending upon the volume of collected whole blood (most often 400-450 ml) and donor's hematocrit. apheresis plasma (also called bsource plasmaq) is collected from donors through a process where blood is removed from the donor, anticoagulated (generally with a 4% sodium citrate solution), 1 and immediately separated by physical means (centrifugation or filtration, or a combination of both) into components. 3 at minimum, the red cells are returned to the donor while plasma is retained and collected in a container (bag or plastic bottle). the duration of a typical plasmapheresis procedure depends on the number of cycles (and, hence, the volume of plasma collected) and lasts generally from 35 to 70 minutes. apheresis plasma volume may range from 450 to 880 ml, depending upon the country's regulations and collection protocol. apheresis plasma can also be prepared as a byproduct of plateletpheresis (bconcurrent plasmaq), a procedure used primarily for the collection of platelets. both recovered and apheresis plasmas are suitable for the manufacture of the whole range of fractionated plasma products. the mean content in coagulation factors, more particularly fviii, is lower in recovered than in apheresis plasma because of (a) longer processing time before freezing (whole blood must be further processed to separate cellular components and plasma), (b) higher ratio of anticoagulant and, possibly, (c) the higher level of cellular contamination that may release proteolytic enzymes affecting the stability of coagulation factors. apheresis plasma contains less igg when collected from frequent donors. protein content and quality of fractionated proteins is apparently not affected by the apheresis system used, although residual cell content differ based upon the type and configuration of the cell separation device. 4 plasma from membrane apheresis procedures, as does recovered plasma prepared from whole blood leukoreduced on positively charged filters, may 4performed by most fractionators. ymandatory in europe for hcv. zmay contribute to viral clearance but does not necessarily result in robust and consistent removal. §for small viruses, robust removal is achieved by narrow pore size membranes (v20 nm). texpected contribution based on experimental studies using spiked tse agents, in the absence of information of the biological nature of the tse-human plasma associated agent. contain more activated complement component 3 and 5 (c3a, c5a) anaphylatoxins, 5-7 but the impact on the quality or yield of fractionated products is unknown. various infectious agents have been identified as potential contaminants of human blood. 8 bacteria, parasites, and intracellular viruses are not transmitted by plasma products because they are destroyed by freeze-thaw steps or removed by the 0.2-to 1-lm filtration steps used during the processing of fractionated products. pathogenic plasma-borne viruses include hiv, hcv, hepatitis b virus (hbv), west nile virus (wnv), hepatitis a virus (hav) and parvovirus b19 (b19). the various complementary safety nets in place during the production chain of fractionated products, from donor selection to industrial product extraction, to optimize safety against these agents are summarized in table 1 . the importance of viral testing on the safety of plasma products has been reviewed. 1, 9, 10 the extent of viral testing of plasma for fractionation takes into account the ability of validated fractionation processes to eliminate viral risks. 11 some testing is performed by blood establishments, other by plasma fractionators (table 1) . individual plasma donations must be negative for anti-hiv 1 and 2, anti-hcv, and hbsag. genomic assays of plasma minipools for nonenveloped hav and b19 may be performed. 11,12 relevance of testing for the absence of hiv p24ag or wnv nucleic acid testing (nat), which may be justified for the safety of non-virally inactivated blood components, is arguable for plasma for fractionation subjected to robust viral reduction steps of enveloped viruses. the industrial manufacturing pool (usually the cryo-poor plasma that is the first homogeneous pooled plasma fraction) is also tested to confirm the absence of serologic and/or genomic viral markers of hiv, hbv, hcv, hav, and b19. in spite of the most rigorous donor screening and donation testing, infectious viruses may still be present in plasma fractionation pools. therefore, the viral inactivation-removal steps that have been deliberately introduced during plasma products manufacture-and that are described below-play a most critical role in ensuring safety. altogether, these overlapping tests should ensure that the viral load of the manufacturing should is both minimal and significantly below the viral reduction capacity of the manufacturing processes used. preserving fviii during blood/plasma collection and preparation is important for most fractionators preparing coagulation factor concentrates. apheresis plasma can generally be frozen quickly, ensuring optimal fviii preservation. by contrast, whole blood has to be centrifuged to separate the various components. when blood is cooled to 48c after collection, plasma should be separated and frozen within 6 to 8 hours to preserve fviii, but when cooled rapidly at constant 208c using devices like butanediol plates, coagulation factors are stable for up to 18 to 24 hours. plasma frozen within 72 hours is suitable for igg and albumin production. 1 after separation from cellular elements, plasma for fractionation should be frozen rapidly below à208c, à258c, or à308c, depending upon local regulations. 13 plasma used to manufacture only albumin and igg may be frozen below à20 8c within 72 hours of collection. 13 in the us code of federal regulations, apheresis plasma should be stored at à208c or colder immediately after collection. rapid plasma freezing, to ensure rapid ice front velocity and core temperature of à208c, preserves fviii and appears more important than the actual freezing temperature itself. 14 plasma for fractionation is stored at less than à20 8c, or colder, typically for several months or more. storage temperature should be as constant as possible, including the transportation to the fractionation facilities. cross-continent or intercontinent shipment of plasma for fractionation is frequent. production steps taking place at fractionation plants are summarized in figure 1 , and typical plasma protein downstream methods are presented in table 2 . physical compliance with shipping requirements is verified at delivery of the plasma frozen plasmas are expelled from the plastic containers and pooled for cryoprecipitation and further manufacturing steps, as described below. intermediate fractions generated during production may be stored for subsequent pooling and processing. purified sterile-filtered products are aseptically dispensed into final containers (glass vials or bottles). albumin bottles undergo terminal pasteurization. many products, but albumin and some igg preparations, are freeze-dried, typically for a duration of 3 to 6 days, depending upon physicochemical characteristics and filled volumes. batches are quarantined while quality controls and checks of production files take place. batches meeting specifications are labeled and packaged and subsequently boxed and shipped for distribution. in a few countries, product batches may be released by regulatory authorities. the production cycle of fractionated products takes a few weeks to several months. current core fractionation technology largely relies on a backbone process encompassing cryoprecipitation and cold ethanol precipitation steps, as developed in the 1940s by cohn et al 15 in the united states, or modified by kistler and nitschman 16 in europe. this process involves successive processing steps at defined ethanol concentrations, associated with shifts in ph, temperature, and osmolality that result in selective precipitation of proteins, most notably igg and albumin. precipitates are separated by centrifugation or filtration. in the last few years, the complexity of the fractionation process has increased by (a) the introduction of chromatography to isolate new proteins from existing fractions such as cryoprecipitate, cryo-poor plasma, and cohn fractions; (b) the integration of chromatography to the ethanol fractionation process to increase igg recovery; and (c) the implementation of dedicated viral inactivation or removal steps. chromatography was introduced in the 1960s; however, its application developed mostly in the mid/late 1980s. anion-exchange chromatography and affinity chromatography are frequently used to capture proteins at physiological ph and ionic strength, therefore best preserving functional activity. 17, 18 immobilized heparin and monoclonal antibodies are common affinity chromatography ligands. chromatography is used for 4 specific goals: (a) improvement of products purity, (b) extraction of trace labile proteins, (c) optimization of protein recovery, and (d) removal of viral inactivation agents. figure 2 illustrates a typical industrial fractionation scheme of standard plasma. plasma packs (typically for a batch of 2000-4000 l) are opened under hygienic conditions, and plasma is expelled from the containers and thawed at 18c to 48c. cryoprecipitate is isolated using refrigerated continuous centrifuges, recovered from the centrifugation bowls and frozen at à308c or colder for storage until further pooling and processing. the cryo-poor plasma is immediately processed for primary chromatographic capture of labile coagulation factors (such as the factor ix [fix] complex and its components) and protease inhibitors (such as at and c1 esterase inhibitor [c1-inh]). the prepurified intermediates may be stored frozen until further processing. the coagulation factors/ anticoagulant-depleted plasma undergoes sequential ethanol precipitation steps. this leads to successive precipitations of fibrinogen, igg and albumin fractions, and intermediates for extraction of other therapeutic proteins, such as aat (fraction iv-1), or igm (fraction iii). depth filtration is preferred to centrifugation to separate precipitates and improve protein recovery. the fractionation of hyperimmune plasma (eg, anti-rhesus) is usually performed on small plasma batch sizes, increas ingly using full chromatographic processes to optimize the recovery of igg. no documented transmission of hiv, hbv, or hcv by products subjected to dedicated viral inactivation treatments has been recorded since the end of the 1980s. 19 viral reduction treatments include inactivation steps (where viruses are bkilledq) and removal steps (where viruses and proteins partition into distinct fractions). use of one, or preferably two, distinct dedicated viral reduction treatments is the current bgold standardq for all plasma products. the first treatment is performed primarily to inactivate the most pathogenic viruses (hiv, hbv, and hcv), whereas the second reduction step targets nonenveloped viruses but also contributes to added safety against all agents. most viral reduction treatments are integrated with the protein fractionation process (bin-processq treatments), but some currently based on heat inactivation procedures are applied on products filled in their final container (terminal treatment). fractionators are required by regulatory authorities to conduct down-scale experimental validation studies using relevant model viruses to establish the efficacy and robustness of viral reduction procedures. the robustness of the viral reduction procedures in place is exemplified by the absence of transmission of wnv, an emerging virus. 20 similarly, the lipid-enveloped severe acute respiratory syndrome coronavirus has been shown to be inactivated by core viral inactivation treatments of plasma products. 21,22 it is also likely, but not proven by validation experiments yet, that avian flu and simian foamy viruses, which also have a lipid envelope, would be inactivated by current processes in place if present in plasma. details on viral validation procedures can be found elsewhere. 23, 24 the major characteristics of current viral reduction treatments are summarized in table 3 9,23 and discussed briefly here. in-process viral inactivation treatments. the solvent-detergent (sd) treatment, developed in the mid 1980s, 25 remains the most frequent core viral inactivation procedure of plasma products. protein solutions are incubated for 4 to 6 hours at 248c to 378c in the presence of 0.3% to 1% tri-n-butyl phosphate (tnbp) and 1% tween-80 or triton x-100. typically, lipid enveloped viruses are inacti-vated in a matter of minutes, and the functional activity of even the most labile plasma proteinswith the possible exception of some serine prolease inhibitors-is well preserved, but nonenveloped viruses (less pathogenic in most individuals) are not inactivated. the sd agents are removed down to a level of a few parts per million usually by chromatographic adsorption or specific precipitation of proteins, or selective adsorption on hydrophobic chromatographic support. pasteurization, another common viral inactivation procedure, is a heat treatment of protein solutions for 10 hours at 608c, a treatment that denatures viral proteins and inhibits virus replication. 26 pasteurization can inactivate both enveloped and nonenveloped viruses, but stabilizers, needed to limit loss of protein functionality, may decrease the rate and extent of viral inactivation. 27 stabilizers may be removed by ultrafiltration, protein precipitation, or chromatography. vapor heat has also been used by one company; extent of virus inactivation is influenced by the temperature, duration, and pressure during treatment. risk of neoantigen formation, which can enhance protein immunogenicity, should be considered when using heat-based inactivation processes. low ph incubation, usually at ph 4, at 308c to 378c for more than 20 hours, was introduced in the early 1980s to allow the intravenous infusion of igg. this form of treatment was subsequently found to inactivate most lipid-enveloped viruses. caprylic (octanoic) acid precipitation/incubation at ph below 6 is a recently introduced treatment of human igg that can inactivate lipid-enveloped viruses. 28, 29 terminal viral inactivation treatments. in modern plasma fractionation, heat treatment of lyophilized products (dry heat) is used, due to limitations, mostly as a secondary viral inactivation step rather than the core inactivation treatment. the treatment is applied to some coagulation factor concentrates. performed at 808c for 72 hours or at 1008c for 30 minutes, generally in the presence of protein stabilizers, it provides added safety against havand other heat-sensitive viruses but may not be sufficient to exclude b19 transmission. 23 terminal (liquid) pasteurization at 608c for 10 hours is the bgold standardq treatment of albumin preparations. the fatty acids, caprylate, and tryptophanate, which protect albumin from heat denaturation, are added at doses compatible with therapeutic use and, therefore, are not removed before product infusion. viral removal treatment. nanofiltration is a specific viral filtration process applied to protein solutions using 15-to 75-nm multi-layers membranes, or equivalent systems, to remove viruses mostly by a sieving mechanism. 30,31 introduced in the early to mid 1990s, it has reached wide acceptance as a robust viral removal step for essentially all products, apart from albumin. nanofiltration is used to complement the core viral inactivation treatment and to provide enhanced safety against nonenveloped viruses or other resistant infectious agents. virus removal can also incidentally take place during protein precipitation, chromatography, or filtration steps; these steps contribute to the lowering of virus load from the protein production stream; they are difficult to monitor; and therefore do not guarantee, as standalone procedures, sufficient safety margin. prion removal methods. variant creutzfeldt-jakob disease (vcjd) can be transmitted by red blood cell concentrates, but to date, transmission has not been identified from plasma or plasma products. because of its biological nature, the prion agent is thought to be resistant to current viral inactivation procedures used during plasma fractionation. the methods known to inactivate abnormal misfolded prion proteins associated with transmissible spongiform encephalopathies (prp tse ) (such as oxidation, treatment with strong base, chaotropic agents, and extreme heat) destroy plasma proteins and, therefore, cannot be used. still, modern processes of fractionated products would seem to ensure significant removal of prp tse , as suggested by experimental spiking studies. 32 as scale-down and experimental transmissible spongiform encephalopathy (tse) spiking models are developed, knowledge on the capacity of manufacturing processes of plasma proteins to remove prions is growing rapidly, although much uncertainty remains because the unknown biological nature of the human plasma associated infectious agent. 33 several processes for manufacturing fviii, fibrinogen, von willebrand factor (vwf), fix, igg, and albumin products have been shown to be capable of removing prions in a consistent and reproducible manner. [34] [35] [36] generally, multistep fractionation processes are thought to be a contributing factor to prp tse elimination. two to more than 5 log removals of spiked prions occur during standard protein purification steps such as precipitation with ethanol or polyethylene glycol (peg), anion-exchange chromatography, and depth filtration. 34, 35, 37 mechanism of removal that may encompass an adsorption mechanism is still not fully understood and appears to be influenced by ph and the concentration of the precipitating agent. the partitioning process may reflect some prion aggregation because of prion hydrophobicity and insolubility. the removal capacity of nanofiltration membranes with pore sizes less than 75 or 35 nm has been extensively investigated, 34, 38 and prion removal is likely due in part to molecular sieving. extent of removal is related to the pore size of the filters and, presumably, to the aggregation state of prp tse . removal is superior with membrane of pore sizes of 15 nm, compared to 35 nm. the nature and origin of the spiking agent used for prp tse clearance studies is of critical importance because various spikes differ in size and characteristics. brain homogenate or brain-derived microsomal fractions from infected animals have usually been used as the source of prp tse spiking material. the most reliable and quantitative detection method of prp tse is based on animal bioassays, which require many animals and a time frame generally longer than 9 months. in vitro immunochemical methods, such as a western blot assay, are being used, at least as a first marker of the presence of prp tse and associated infectivity, by detecting the protease-resistant fragment. 33 thus, the risk transmission of vcjd by human plasma products appears remote, but caution should prevail since the biochemical nature of the infectious agent in human blood is not known. technologies to extract coagulation factors, protease inhibitors, and igg have evolved considerably in the last 20 years, leading to the development of products with improved safety and purity profiles. a description of major manufacturing techniques is given below. factor viii. several generations of fviii preparations have been developed since the mid-1980s, providing (a) safety from hiv and then hcv and hbv, (b) improved purity, and (c) enhanced safety from hav and b19. current development efforts focus on establishing prion removal. 36 all currently licensed plasma-derived fviii concentrates are purified from cryoprecipitate. in a typical process, cryoprecipitate is subjected to a combination of aluminium hydroxide adsorption and precipitation, or precipitation only (eg, using glycine), to reduce the level of trace vitamin k coagulation factors (as they may activate fviii during the downstream purification steps) or load proteins such as fibrinogen. the purified cryoprecipitate extract usually undergoes viral inactivation typically by sd or pasteurization. many processes include subsequent chromatography by anion exchange, monoclonal antibody affinity (using anti-fviii or anti-vwf murine antibodies), or immobilized heparin affinity to remove protein contaminants (such as fibrinogen or fibronectin), most or part of the vwf, and the sd agents. 39 immunopurified fviii eluate is further purified by chromatography to remove murine igg ligands that may have leached. prior to formulation and sterile filtration, some fviii products are nanofiltered using membranes with a pore size of 35, 20, or even 15 nm if a partial dissociation of fviii and high-molecular-weight vwf multimers is initiated. alternatively, some freeze-dried preparations are subjected to heat treatment at 808c or 1008c to inactivate nonenveloped viruses like hav. recovery of fviii, as expressed per liter of plasma, is usually comprised between 100 and 200 iu (1 iu is defined as the physiological activity present in 1 ml of plasma). factors lowering fviii yield include cryoprecipitation (ca 30% loss), chromatographic purification (ca 20% to 30 %), and viral heat inactivation (15%-30%). current fviii concentrates have a specific activity between 10 and 250 iu/mg. some products are formulated with human plasma-derived albumin, whereas others contain copurified vwf that helps stabilize fviii. purity of fviii concentrates has not been convincingly demonstrated to enhance immunological safety. long-term clinical experience indicates that the alleged reduced immunosuppressive effects of immunopurified preparations compared to lowerpurity plasma-derived preparations, as claimed in the late 1990s, were probably unfounded. processes used that, in part, influence residual vwf, may have an impact on the immunogenicity of plasma-derived fviii products. retrospective studies of previously untreated patients show that an sd-treated, nanofiltered, ion-exchange purified fviii product containing vwf appears to be 2 times less likely to induce anti-fviii inhibitors in hemophilia a patients than 2 full-length recombinant fviii concentrates. 40 von willebrand factor. because fviii chromatographic purification removes all, or part, of vwf, fviii products effective in treating von willebrand disease (vwd) are generally low-purity (bintermediate purityq) preparations prepared from cryoprecipitate by precipitation steps coextracting vwf and fviii in a ratio of higher than 1. the low purity and the high protein content of these products technically restrict the choice of viral inactivation treatments to pasteurization or terminal dry heat at 808c for 72 hours. one highly purified vwf concentrate, largely devoid of fviii and, therefore, specific for vwd treatment, is prepared from cryoprecipitate by a 3-step chromatographic procedure (integrated with fviii and fibrinogen purification processes) using 2 anion exchangers and immobilized gelatin polishing (to remove fibronectin). 41 viral reduction is by sd, 35-nm nanofiltration, and terminal dry heat at 808c for 72 hours. 42 fibrinogen. there are 5 registered fibrinogen preparations available for treating a fibrinogenemia or hypofibrinogenemia. 43 traditional preparations are obtained by multiple precipitation steps of plasma or cryoprecipitate using ethanol and glycine, whereas other modern products are purified by chromatography. viral reduction is achieved by sd treatment, often complemented by 35-nm nanofiltration or terminal dry heat treatment. single-step pasteurization at 608c for 20 hours is used for 1 product. fibrin sealants. fibrin sealants (fibrin glues) comprise fibrinogen-rich and purified thrombin concentrates. when mixing the 2 components, a strong adhesive clot exhibiting hemostatic, sealing, and healing properties is formed almost instantaneously or within a few seconds, offering multiple topical surgical applications. fibrinogen is prepared by precipitation methods from cryoprecipitate, or from the cohn fraction i; the fraction may also contain fibronectin, vwf, or factor xiii (fxiii), which may confer other physiological functions. 44 fibrinogen fractions are virally inactivated by sd, pasteurization, vapor-heat treatment, and/or nanofiltration. the fibrinogen concentration is typically above 80 g/l and may be formulated in the presence of an antifibrinolytic agent. prothrombin complex. prothrombin complex concentrate (pcc) is a mixture of vitamin kdependent coagulation factors in which fix, factor ii, and factor x and proteins c and s have a low specific activity between 0.5 and 2 iu/mg. 45 a few products contain also factor vii (fvii), but usually at levels lower than that of fix. the manufacture is usually based on a 1960s method that involves diethylaminoethyl (deae) sephadex or deae cellulose adsorption of cryo-poor plasma, but downstream ethanol fractions can also be used as starting materials. 46 anion exchangers coextract proteins sharing the presence of gamma-carboxyglutamic acid residues, whereas bulk plasma proteins, such as albumin and igg or at and aat remain in the unbound fraction. precipitation with tricalcium phosphate is used for one product. viral reduction is most often achieved by sd (tnbp-tween 80) treatment, complemented by 35-or 15-nm nanofiltration or by terminal dry heat. pasteurization and vapor heat are applied to 2 products. viral inactivation treatment by sd requires one subsequent ionexchange chromatographic step for removal of the virus-inactivating agents. recovery is in the range of 250 to 380 iu of fix per liter of plasma. single fix. high-purity fix products were developed in late 1989, 47 leading to reduced risks of thromboembolism, compared to pcc, in hemophilia b patients. fix is isolated by chromatographic purification of the pcc using anion exchange combined with either immobilized heparin, metal chelate affinity, or monoclonal antibody. these processes yield fix concentrates with a mean specific activity in the range of 100 to 150 iu/mg and a yield between 200 and 300 iu/l of plasma. factor vii. three specific concentrates rich in fvii and with reduced amount of other vitamin kdependent clotting factors are currently licensed to control bleeding in deficient patients. the manufacturing process includes ion-exchange chromatography or aluminium hydroxide adsorption, following a downstream procedure similar to that of pcc and fix. viral inactivation is achieved by sd treatment, vapor heat, or dry heat. factor xi. two factor xi (fxi) concentrates are currently available for deficient patients. one, of low purity, is purified by chromatography of cryo-poor plasma on deae cellulose and immobilized heparin. after freeze-drying, the product is virally inactivated by dry heat. in the other product, fxi is captured by adsorptive filtration then highly purified by cation-exchange chromatography. 48 this product undergoes dual viral reduction processing by sd and 15-nm nanofiltration. factor xiii. factor xiii is a transglutaminase that catalyzes the final step in the coagulation cascade, cross-linking the loose fibrin polymer into a highly organized structure. the early generation of fxiii concentrates for the treatment of fxiiideficient patients was extracted from placenta, but 2 plasma-derived products have subsequently been developed. one is purified from a cold-ethanol fraction from cryoprecipitate supernatant, purified by precipitation with sodium citrate and removal of fibrinogen by heating. the product is pasteurized in sorbitol solution, ultrafiltered to remove sorbitol, adsorbed with bentonite, and freeze-dried. the other product is obtained by precipitation steps and is also pasteurized. factor xiii is also a component of some fibrin sealants. although still subject to discussion, the presence of fxiii is claimed to contribute to fibrin c-chain cross-linking and tensile strength, possibly improving the hemostatic effect. 49 activated coagulation factors. human thrombin concentrates are available so far only as components of fibrin sealant. thrombin is prepared by activation of the pcc, usually in the presence of calcium chloride, followed by viral inactivation treatment by sd, purification by cation-exchange chromatography, and viral removal by 15-nm nanofiltration. final thrombin concentration is usually between 300 and 1000 iu/ml, but preparations with lower potency are available when a slower speed for clot formation of the sealant is preferred for some surgical applications requiring longer time for tissue gluing. a procedure for large-scale production of a purified plasma-derived fviia has been developed in japan. 50 fvii is purified by anion exchange and immunoaffinity chromatography and converted to fviia by autoactivation on an anion-exchange resin and incubation in the presence of ca 2+ for 18 hours at 108c. this preparation is virally reduced by nanofiltration and dry-heating and is intended for the treatment of hemophiliacs with antibodies against fviii or fix. antithrombin. antithrombin concentrates were the first plasma products extracted by affinity chromatography. production from cryo-poor plasma usually comprises ion exchange chromatography to remove the pcc components, followed by capture of at on immobilized heparin. viral inactivation is traditionally achieved by pasteurization in the presence of sodium citrate or a combination of sucrose and glycine, although sd treatment is used as well. because heat treatment may partially denature at, a second adsorption step on immobilized heparin can be used to remove altered molecules. recovery is between 250 and 350 u/l of plasma. fraction iv-1 is an alternative starting material, but yield is significantly lower. a1-antitrypsin (a1-protease inhibitor). there are now several licensed aat concentrates, including 3 in the united states. a1-antitrypsin augmentation therapy is indicated for the treatment of patients with lung emphysema secondary to congenital aat deficiency. because aat shares many physicochemical properties, in particular, molecular weight and isoelectric point, with albumin, it has been difficult to design production methods for aat not affecting the existing production process for albumin. most preparations are recovered from fraction iv [1] [2] [3] [4] . purification from this waste fraction is rather cumbersome and involves peg precipitation and ion-exchange chromatography, considerably compromising recovery (~0.2 g/l). the first preparations developed in the 1990s were virally inactivated by pasteurization, but dual viral reduction steps using sd and nanofiltration are also now used. recent isoelectrofocusing studies have provided evidence indicating that new anodal aat variants are present in at least one of the fda-licensed product, suggesting alteration of some isoforms during purification. loss of a c-terminal positive charged lysine, secondary to carboxypeptidase activity, was proposed as an explanation for these isoelectrofocusing migration shifts. under circumstances when clinical demand for albumin decreases, extracting aat from upstream fractions, such as the supernatant ii+iii, 51 appears a logical trend, offering the possibility of a more effective production scheme, characterized by a recovery of 0.6 to 1 g/l. 51 protein c. there are 2 protein c concentrates manufactured in europe and 1 in japan. in one process, the pcc undergoes cascade purification on 3 ion exchangers, 52 whereas in the other, immunoaffinity and affinity chromatographic processes are combined with ion exchange. viral reduction is achieved by sd treatment, which can be combined with 15-nm nanofiltration or vapor-heat treatment. 53 c1-esterase inhibitor. c1-esterase inhibitor concentrates are used for the treatment of acute phases of angioedema, primarily in the oropharyngeal region and gastrointestinal tract in patients with congenital or acquired c1-inh deficiency. there are 3 products licensed in europe. products are generally purified by chromatography from the cryo-poor plasma after extraction of the pcc and, potentially, at. viral inactivation is achieved by pasteurization, vapor heat, or sd, possibly combined with nanofiltration. essentially all therapeutic albumin preparations are prepared by fractionation of cryo-poor (or pccpoor and/or at-poor and/or c1-inh-poor) plasma by ethanol fractionation. a critical upstream process step (precipitation ii+iii) separates the igg fraction. to optimize recovery, the precipitates generated during the ethanol fractionation process are separated by depth filtration. albumin recovery of 75% to 85% (25-28 g/l) and purity of 96% to 98% are typically obtained. some processes combine ethanol fractionation with a polishing ion exchange chromatography, which generally improves product purity to ca 99%, whereas in one production method, albumin is purified mostly by anion exchange, cation-exchange, and size-exclusion chromatography. the adjustment of the concentration of the purified fraction, typically from 4% to 25%, is achieved by ultrafiltration. the standard viral inactivation method is pasteurization, which, according to most pharmacopeias, should be performed in the final container rather than on the albumin batch before aseptic filling. current mean albumin yield is 24 to 26 g/l of plasma. polyvalent igg preparations, either for intramuscular or intravenous uses, are traditionally prepared from the fraction ii that is obtained by stepwise fractionation of cryo-poor plasma using cold ethanol at concentrations up to 25%. increasingly, igg products are extracted from up-streamed ethanol precipitated fractions, such as supernatant iii or precipitate ii+iii, to optimize recovery. intermediate igg fractions are subjected to ion-exchange chromatography, caprylic acid, or peg precipitations to remove protein contaminants, proteolytic enzymes, and/or aggregates. most current viral inactivation procedures are low ph incubation, pasteurization, or sd; the caprylic acid treatment, recently introduced in the manufacture of human igg products, is also a robust viral inactivation process of igg. dedicated viral removal by 15-to 35-nm nanofiltration is commonly used to increase the safety against nonenveloped viruses, especially in a situation when the core viral inactivation treatments target only lipid-enveloped viruses. the igg recovery has long been in the 2.7-to 3.2-g/l range when combining traditional ethanol fractionation processes and centrifugation. depth-filtration and/or chromatographic purification from upstream fractions have improved the mean recovery to the 3.5-to 4.5-g/l range, or more. total chromatographic procedures are increasingly used for the production of hyperimmune igg products because such processes are amenable to the fractionation of smaller plasma volumes and can optimize recovery. the manufacturing process includes at least 2 dedicated viral reduction treatments. other protein components have been fractionated at pilot-scale and subjected to experimental or human trials. inter-a-trypsin inhibitor (iti) is a kunitz-type serine proteinase inhibitor. its inhibitory capacity is carried by bikunin, a chondroitin 4-sulfate proteoglycan, which is covalently linked to its heavy chains h1 and h2, but can be released by proteolytic cleavage. an iti concentrate has been obtained by fractionation of the prothrombin complex on an anion exchanger followed by immobilized heparin and viral inactivation by sd. iti, as a reservoir of bikunin, may be involved in control of inflammatory processes. in a porcine model of endotoxin shock, iti improved the hemodynamic, oxygenation, and coagulation parameters. 54 administration of iti very early after the onset of sepsis or repeated injections at later time points (10 and 20 hours) maintains cardiovascular stability and significantly reduces mortality in a rat model. 55 transferrin is the major iron binding plasma protein which may prevent cytotoxic effects or predisposition to septic infection due to accumulated free non-transferrin-bound iron when normal iron use is hampered and/or apotransferrin production is decreased. a liquid apotransferrin concentrate has been obtained from cohn fraction iv by 2 ion exchange chromatographic steps and ultrafiltration. viral safety was ensured by sd treatment, nanofiltration, and peg precipitation. the product had intact iron binding capacity, and maintained the bacterial growth inhibitory effect in serum. in hematological stem cell transplant patients, the product prevented the appearance of non-transferrin-bound iron. apolipoprotein a-i is the principal protein component of the plasma high-density lipoproteins. it prevents the accumulation of cholesterol-loaded macrophages which deposit on the arterial wall as foam cells. apolipoprotein a-i inhibits hepatic lipase and lipoprotein lipase in vitro. a concentrate has been isolated by precipitation from cohn fraction iii. 56 intravenous injections in men were well tolerated in an early clinical trial; clinical observations were consistent with combined inhibition of hepatic lipase and lipoprotein lipase activities. possible clinical applications include the treatment of hypercholesterolemic patients and atherosclerosis. recombination with lecithin forms a high-density lipoprotein complex that could help limit inflammation, endotoxin-induced activation of coagulation, and fibrinolysis in septic conditions. mannan-binding lectin (mbl) is a component of the innate (aspecific) immune system that can bind repetitive structures of mannan groups, such as those on the surface of micro-organisms, activating the complement system and leading to the destruction of a large variety of micro-organisms. the relatively frequent congenital deficiency of mbl is associated to recurrent infections, especially in infants when the specific immune system has not yet matured. an mbl concentrate has been produced from cohn fraction iii. 57 plasmin is the major fibrinolytic enzyme in plasma. encouraging results as a new fibrinolytic agent have been obtained in animal models where plasmin was applied directly to the clot through a catheter to treat peripheral arterial occlusion. von willebrand factor cleaving protease (vwf-cp, adamts13) cleaves ultra-large multimers of vwf that enter the blood stream directly after biosynthesis by endothelial cells. if developed, a purified vwf-cp could be useful to treat, in place of plasma, patients with thrombotic thrombocytopenic purpura with congenital or acquired deficiency of vwf-cp. activated protein c can be of value for the treatment of sepsis, as demonstrated through the clinical use of a recombinant preparation. there is no therapeutic plasma-derived activated protein c products available yet for therapeutic use, although a process for a highly purified preparation, where cryo-poor plasma is purified by immunoaffinity and anion-exchange chromatographic steps, and sd viral inactivation has been described. 58 there is also research needed to investigate alternative indications for currently available products. potential clinical use of c1-inh, aimed at benefiting from its role as inhibitor or attenuator of the activation of complement and contact systems, include septicemia, myocardial infarction, capillary leak syndrome, pancreatitis, and organ transplantation. intravenous use of a fxiii concentrate was found to increase epidermal growth factor and transforming growth factor-b, suggesting that it may accelerate wound healing of anastomotic leaks and nonhealing fistulas. factor xiii was also found to have osteoinductive properties, suggesting use in bone tissue engineering. a review on plasma fractionation should also cover the role played by national and international regulatory authorities. over the last few years, assuring the safety of large-pool plasma products has posed formidable challenges to regulatory authorities and fractionators alike. the complexity of the field, encompassing the diversity in blood and plasma product types and manufacturing processes, made it difficult to enact balanced decisions toward ensuring both product safety and guarantee of supply. since the 1980s, the agencies regulating the plasma fractionation industry have developed a comprehensive set of measures to ensure the viral safety of plasma products. multiple layers of regulatory oversight of the plasma industry have been established to ensure overlapping safeguards against the risks of the transmission of bloodborne infectious agents. several regulations, guidances, position statements have been issued by agencies like the us fda and the european medicine evaluation agency, which are updated as needed. those cover important safety aspects required at all stages of the manufacturing chain, from activities at blood establishment preparing plasma for fractionation, extending to the manufacturing and distribution of plasma products. regulatory oversight includes epidemiologic surveillance of the donor population; donor deferral policies and screening practices; mandatory donation testing; testing of manufacturing plasma pools; validation of viral reduction procedures and other production steps; as well as the assessment of product quality, safety, and efficacy for marketing authorization. most of relevant information on plasma collection is assembled in europe in the plasma master file, which allows establishing key levels of information regarding the quality and safety of the plasma raw material. post marketing, reports on adverse reactions associated with plasma products should be transmitted to nras and could prompt emergency response procedures and product recalls. 10 some harmonization of the requirements for manufacture and supply of plasma products in the united states, europe, and japan is taking place under the auspices of the international conference on harmonisation, but much work remains to be done. such measures, nonetheless, provide the framework through which modern plasma products now exhibit a very high level of quality, safety, and efficacy. however, the rigidity of the regulatory system has been an impediment to more significant technological evolution of the plasma fractionation process because process changes are currently associated to major regulatory work. the cohn plasma fractionation method initially designed to obtain albumin has, over the years, developed rather successfully into a well-established industrial procedure isolating a wide range of clinically useful products. today, more than 20 different protein products, and more if one considers the variety of hyperimmune igg preparations, can be extracted through large-scale fractionation of human plasma. the soundness and large-scale adaptability of the technology, on the one hand, and the rigidity of the current regulatory framework, on the other, explains why this technology remains the main core method in use at industrial scale, although it implies suboptimal yield for most proteins, apart from albumin. the technology has increased in complexity over the years, with the greatest progresses in purification being, without doubt, associated with the use of chromatographic methods that have made possible the development of new protein therapeutics and impressive improvements in product purity and quality. the fractionation scheme has also changed dramatically through the introduction of in-process viral reduction treatments, which have required the addition of downstream techniques such as chromatography and ultrafiltration. the change in protein drivers, with the prominent clinical role now played by igg, and the requirements to increase protein recovery and optimize the fractionation process may crystallize the incentive for most fractionators to abandon relative technical conservatism and introduce significant technological changes in processing technology. the production of igg from precipitate (i)+ ii+iii, already implemented by some manufacturers and, possibly, that of aat from supernatant (i)+ ii+iii, are signs of a gradual evolution in the direction of total chromatographic processing. with so much gained, over the last few years, in the understanding of the key parameters building plasma product quality, safety, and efficacy, one can hope that the regulatory paths, in particular, with regard to clinical studies, to license known protein therapeutics prepared by improved, more efficient technology should be simplified. this would, in turn, contribute to an improved supply. as the plasma fractionation industry has so far been targeting mostly proteins that were obvious candidates for replacement therapy, it is also hoped that it will invest more into developing new products because plasma remains a unique source of potential therapeutic proteins. proactive research and development work should be encouraged to isolate and evaluate new therapies among the well characterized plasma proteins with still unknown function. finally, one should not forget that plasma protein therapies are expensive and largely inac-cessible to the developing world. the new market drivers in rich countries are likely to diminish economical interest in the manufacture of fviii that remain a major protein therapy in need in the developing world. the belief that decreased use of plasma-derived fviii or fix in developed economies (as hemophiliacs are switching to recombinant therapies) would increase the supply of products to developing countries may be not economically viable. rather, this switch to recombinant products in rich countries can make poor countries unable to afford the increased cost of products no longer subsidized by the premium price paid in rich countries. it therefore remains important to develop affordable viral inactivation and processing technologies gradually allowing developing countries to make use of local plasma resources in a safe manner. 59 guideline on epidemiological data on blood transmissible infections. for inclusion in the guideline on the scientific data requirements for a plasma master file current instrumentation for apheresis. apheresis: principles and practice assessment of complement activation during membrane-based plasmapheresis procedures the effect of leukocyte depletion on the quality of fresh-frozen plasma current safety of the blood supply in the united states farrugia a: guide for the assessment of clotting factor concentrates for the treatment of hemophilia. www.wfh.org. montreal, world federation of hemophilia preparation and properties of serum and plasma proteins. iv. a system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids eight years experience with the alcohol fractionation procedure of nitschmann, kistler and lergier tabor e: the epidemiology of virus transmission by plasma derivatives: clinical studies verifying the lack of transmission of hepatitis b and c viruses and hiv type 1 contribution and interpretation of studies validating the inactivation and removal of viruses (revised) inactivation of viruses in labile blood derivatives. i. disruption of lipidenveloped viruses by tri(n-butyl)phosphate detergent combinations pasteurization of antihemophilic factor and model virus inactivation studies enveloped virus inactivation by caprylate: a robust alternative to solventdetergent treatment in plasma derived intermediates evaluation de l'efficacité des procédés de purification des proteins plasmatiques à éliminer les agents transmissibles non conventionnels distribution of a bovine spongiform encephalopathy-derived agent over ionexchange chromatography used in the preparation of concentrates of fibrinogen and factor viii al: influence of the type of factor viii concentrate on the incidence of factor viii inhibitors in previously untreated patients with severe hemophilia a in vitro study of a triple-secured von willebrand factor concentrate fibrin sealant: scientific rationale, production methods, properties, and current clinical use properties of a highly purified human plasma factor ix:c therapeutic concentrate prepared by conventional chromatography large-scale production and properties of human plasma-derived activated factor vii concentrate effects of inter-alpha-inhibitor in experimental endotoxic shock and disseminated intravascular coagulation delayed administration of human inter-alpha inhibitor proteins reduces mortality in sepsis a minipool process for solvent-detergent treatment of cryoprecipitate at blood centres using a disposable bag system key: cord-018845-r88bhiac authors: sachs, u. j. h.; bux, j. title: gewinnung, herstellung und lagerung von blut und blutkomponenten date: 2010-11-21 journal: transfusionsmedizin und immunh&#x000e4;matologie doi: 10.1007/978-3-642-12765-6_16 sha: doc_id: 18845 cord_uid: r88bhiac blutspender leisten einen wertvollen dienst für die gemeinschaft: die ständige verfügbarkeit von blutkomponenten ist zur unverzichtbaren voraussetzung für viele bereiche der medizin geworden. nicht nur die gewinnung und aufarbeitung von blut und blutbestandteilen zur sicherstellung einer qualitativ wie quantitativ guten versorgung, sondern auch die kompetente betreuung der spender ist eine der großen aufgaben der transfusionsmedizin. blutspender leisten einen wertvollen dienst für die gemeinschaft: die ständige verfügbarkeit von blutkomponenten ist zur unverzichtbaren voraussetzung für viele bereiche der medizin geworden. nicht nur die gewinnung und aufarbeitung von blut und blutbestandteilen zur sicherstellung einer qualitativ wie quantitativ guten versorgung, sondern auch die kompetente betreuung der spender ist eine der großen aufgaben der transfusionsmedizin. der verlust von bis zu 15 % des blutvolumens über einen zeitraum von etwa 5 min führt in der regel nicht zu klinischer symptomatik. zwar sinkt der venöse druck leicht ab und erreicht erst rund 30 min später wieder den ausgangswert, blutdruck und pulsfrequenz bleiben jedoch unverändert. eine erhöhte vasokonstriktion und die mobilisierung von blut aus den venösen anteilen des gefäßsystems sichern die blutmenge, die zur erhaltung des normalen herzschlagvolumens erforderlich ist. da männer pro kg körpergewicht (kgkg) durchschnittlich 75 ml blut haben, frauen durchschnittlich 66 ml blut, können bei gesunden spendern mit mehr als 50 kgkg bis zu 525 ml blut (einschließlich untersuchungsproben) entnommen werden. die klinische erfahrung zeigt, dass selbst die entnahme von 1 l blut häufig zu keiner veränderung des blutdrucks führt, solange der spender liegt. mit dem aufrichten kann es allerdings zur beeinträchtigung der kreislauffunktion und zu klinischer symptomatik kommen. ein verlust von 1500-2000 ml blut führt dann zu blutdruckabfall und vermindertem kardialem schlagvolumen mit der subjektiven empfindung von kälte und atemnot. neben der kompensatorischen vasokonstriktion kommt es auch zum zustrom von interstitieller flüssigkeit in das gefäßsystem. der durch die blutspende bedingte volumenverlust wird durch eine erhöhung des plasmavolumens innerhalb von 24 h ausgeglichen. dieser volumenzustrom bedingt gemeinsam mit der allgemeinen stressreaktion als antwort auf die blutspende eine veränderte zusammensetzung des peripheren blutes. die gesamtleukozytenzahl steigt um 25 % gegenüber dem ausgangswert an, wobei die zahl der eosinophilen, der lymphozyten und der monozyten mäßig absinkt. diese reaktion erreicht ihr maximum 2-3 h nach der spende und klingt nach etwa 5 h ab. auch die thrombozytenzahlen sinken zunächst um 10.000-15.000/μl ab, um nach wenigen stunden wieder die ausgangswerte zu erreichen. erythrozytenzahl und hämatokrit zeigen nach 4-5 h eine sinkende tendenz und erreichen nach etwa 24 h die niedrigsten werte; im mittel sinkt die erythrozytenzahl um 250.000-350.000 × 10 9 /l, der hämoglobinwert um 1 g/dl und der hämatokrit um 3 %. der abfall des hämoglobins verschiebt die o 2 -dissoziationskurve nach rechts und verbessert dadurch die o 2 -abgabe an das gewebe. die leichte hypoxämie stimuliert die bildung von erythropoetin und somit die erythropoese. diese geringfügigen veränderungen des roten blutbildes sind für den gesunden erwachsenen ohne bedeutung: spätestens nach einer woche sind die erythrozyten vom knochenmark ersetzt worden. die qualität der neu gebildeten erythrozyten ist dabei natürlich von den eisenreserven des organismus abhängig. verminderte eisenreserven sowie ein verzögerter ausgleich des hämoglobins und des erythrozytenverlustes infolge eines latenten eisenmangels finden sich dabei häufiger bei frauen als bei männern. der einfluss einer blutspende auf den eisenhaushalt ist erheblich: der durchschnittliche eisenverlust beträgt 200-250 mg, der durchschnittliche gesamteisengehalt des menschlichen körpers liegt bei etwa 3500 mg (einzelheiten 7 kap. 9). der durch die spende verursachte verlust an plasmaeiweißen wird im durchschnitt mit 8 % des ausgangswertes angegeben. er wird praktisch sofort ausgeglichen, da der organismus über eine ausreichende eiweißreserve verfügt. aufgrund der physiologischen überlegungen sollen im rahmen der blutspende bei erwachsenen, die mehr als 50 kg wiegen, nicht mehr als 500 ml vollblut (zuzüglich untersuchungsproben) entnommen werden. zwischen zwei spenden sollen im regelfall 12 wochen, mindestens aber 8 wochen liegen, und die jährlich entnommene blutmenge darf 2 l bei frauen und 3 l bei männern nicht übersteigen. blutspender leisten einen wichtigen beitrag für die gemeinschaft. gemäß dem ethischen kodex für blutspenden der internationalen gesellschaft für bluttransfusion hat die blutspende freiwillig zu erfolgen, insbesondere finanzieller nutzen darf kein beweggrund sein. hierauf hebt auch §10 des transfusionsgesetzes ab, in dem es heißt: »die spendeentnahme soll unentgeltlich erfolgen. der spendenden person kann eine aufwandsentschädigung gewährt werden, die sich an dem unmittelbaren aufwand je spende orientieren soll.« in deutschland werden blutspenden von regionalen blutspendediensten, die oft kliniknah tätig sind, und von den überregionalen blutspendediensten entnommen. beide ergänzen sich in ihrem versorgungsauftrag. blut spenden kann, wer mindestens 18 und höchstens 68 jahre alt ist. auch ältere spender können nach individueller ärztlicher entscheidung zur blutspende zugelassen werden. die spendetauglichkeit wird durch eine vom spender per unterschrift zu bestätigende anamnese, durch die ärztliche untersuchung und durch laboruntersuchungen gesichert. die entscheidung, ob ein spendewilliger zur spende geeignet ist, wird vom arzt getroffen. dabei muss er die in den richtlinien zur gewinnung von blut und blutbestandteilen und zur anwendung von blutprodukten (hämotherapie) der bundesärztekammer [66] festgelegten vorgaben beachten, die in der jeweils gültigen fassung als stand der medizinischen wissenschaft und technik im sinne des transfusionsgesetzes anzusehen sind. vor der blutspende muss der spendewillige über das wesen, die bedeutung und die durchführung der spendeentnahme und der untersuchungen umfassend aufgeklärt werden. neben der aufklärung und einwilligung muss der spendewillige auch die verwendbarkeit seiner spende erklären; diese erklärungen sind schriftlich abzugeben. die spendeentnahme selbst und alle damit verbundenen maßnahmen sind zu protokollieren und mindestens 15 jahre aufzubewahren; auch hierzu muss der spendewillige schriftlich sein einverständnis geben. neben einer unauffälligen organ-, infektions-und suchtanamnese sowie einem subjektiven gesundheitsgefühl müssen folgende voraussetzungen erfüllt sein: körpergewicht mindestens 50 kg, blutdruck systolisch 100-180 mmhg, diastolisch unter 100 mmhg, regelmäßiger puls mit einer frequenz von 50-100/min (bei ausdauersportlern auch weniger), kein fieber und keine erkennbaren krankheitszeichen. bei frauen muss der hämoglobinwert über 12,5 g/dl (oder der hämatokritwert über 38 %) liegen, bei männern sind die grenzwerte 13,5 g/dl bzw. 40 %. auf dauer von der blutspende auszuschließen sind nach den richtlinien alle personen, 5 bei denen eine hcv-, hiv-, oder htlv-i/ii-infektion nachgewiesen wurde, unabhängig davon, ob krankheitserscheinungen aufgetreten sind, 5 die einer gruppe mit einem gegenüber der allgemeinbevölkerung deutlich erhöhten risiko für eine hbv-, hcv-oder hiv-infektion angehören oder dieser zugeordnet werden müssen (insbesondere homo-und bisexuelle männer, drogenabhängige, männliche und weibliche prostituierte, häftlinge), 5 die an einer protozoonose erkrankt sind oder waren (insbesondere malaria, babesiose, trypanosomiasis, leishmaniasis), 5 die an syphilis, brucellose, rickettsiose, lepra, rückfallfieber, tularämie oder anderen, chronisch-persistierenden bakteriellen infektionen erkrankt sind oder waren, 5 die an bösartigen neoplasien leiden oder litten, wobei in-situ-karzinome und basalzellkarzinome nach kompletter entfernung ausdrücklich ausgenommen werden, 5 die alkoholkrank, medikamentenabhängig oder rauschgiftsüchtig oder dessen begründet verdächtig sind, 5 bei denen ein erhöhtes risiko für die übertragung spongiformer enzephalopathien besteht, insbesondere, weil sie mit hypophysenhormonen humanen ursprungs behandelt wurden, kornea-oder dura-mater-transplantate erhalten haben, bei ihnen oder ihrer familie spongiforme enzephalopathien vermutet oder nachgewiesen wurden oder sie sich zwischen 1980 und 1996 länger als sechs monate in großbritannien aufgehalten haben bzw. nach 1980 in großbritannien eine bluttransfusion und/oder operation erhalten haben, 5 die xenotransplantate erhalten haben. dauerhaft von der blutspende ist auch zurückzustellen, wer an chronischen erkrankungen leidet oder litt und bei dem die blutspende eine eigene gefährdung oder eine gefährdung des empfängers nach sich ziehen kann. personen, die ständig mit arzneimitteln behandelt werden, können nach beurteilung durch den arzt zur spende zugelassen werden. ein dauerausschluss gilt auch für personen mit hbv-infektion, es sein denn, die erkrankung liegt mehr als fünf jahre zurück und virologische kriterien sprechen für eine erloschene kontagiosität (z. b. anti-hbs über 100 ie/l und kein nachweis von hbv-genom in einer sensitiven nukleinsäurenachweistechnik). eine zeitlich begrenzte zurückstellung von der blutspende ist in der regel angezeigt, wenn der spendewillige sich einem infektionsrisiko ausgesetzt hat oder einem solchen ausgesetzt wurde. nach den richtlinien [66] 5 nach einreise aus hiv-, hcv-, hbv-oder htlv-i/ii-endemiegebieten, wenn dort der zeitweilige lebensmittelpunkt lag, 5 nach intimkontakt mit personen, die einer gruppe mit erhöhtem infektionsrisiko für hbv, hcv und/oder hiv angehören (insbesondere homo-und bisexuelle männer die besonderheiten bei der gewinnung von hyperimmunplasma sind in einer entsprechenden richtlinie der bundesärztekammer zusammengefasst [65] . neben den für apheresespender festgelegten kriterien ist zu beachten, dass die thrombozyten des spenders nicht durch medikamente in ihrer funktion beeinträchtigt sein dürfen (z. b. durch acetylsalicylsäure). vor der apherese bzw. innerhalb von 15 min nach beginn der apherese ist neben dem hb-wert auch die thrombozytenzahl zu bestimmen; diese muss über 150 × 10 9 /l betragen. die eignungsuntersuchung soll anlässlich der ersten thrombozytapherese und nach jeder 10. thrombozytapherese, spätestens aber nach 2 jahren durchgeführt werden. das maximale entnahmevolumen beträgt 750 ml (einschließlich antikoagulans, zuzüglich untersuchungsproben). pro jahr können bis zu 26 thrombozytapheresen durchgeführt werden, wobei auch tägliche thrombozytapheresen an 5 aufeinanderfolgenden tagen möglich sind; zwischen einem 5-tage-zyklus und der nächsten spende müssen dann 14 tage liegen, ein erneuter 5-tage-zyklus ist erst wieder nach 3 monaten möglich. sollen anlässlich einer erythrozytapherese 2 präparate gewonnen werden, gelten für alle spender untere grenzwerte von 14,0 g/dl hämoglobin sowie ein minimales körpergewicht von 70 kg. für die eignungsuntersuchung gelten dieselben vorgaben wie bei der thrombozytapherese. das maximale entnahmevolumen pro apherese beträgt 500 ml, auch für die entnahme von 2 präparaten. nach der erythrozytapherese müssen mindestens acht wochen bis zur nächsten vollblutspende oder erythrozytapherese vergehen; nach der gewinnung von zwei erythrozytenpräparaten müssen 16 wochen bis zur nächsten vollblutspende oder erythrozytapherese verstreichen. das gesamtspendevolumen darf 1000 ml erythrozyten bei frauen bzw. 1500 ml erythrozyten bei männern pro jahr nicht übersteigen. die gleichzeitige entnahme mehrerer produktarten, z. b. die gleichzeitige gewinnung von einem thrombozytenkonzentrat und einem plasma oder von einem erythrozytenkonzentrat und einem plasma während einer apherese, ist grundsätzlich möglich. die eignung zur multikomponenten-apherese ist anlässlich der ersten spende sowie anlässlich jeder zehnten multikomponenten-apheresespende zu überprüfen, mindestens jedoch im abstand von zwei jahren. da die apheresesysteme eine reihe von kombinationsmöglichkeiten bieten, sollte darauf geachtet werden, dass die multikomponenten-apheresespende den spender nicht stärker belastet als jede einzelspendeart. das maximale bruttoentnahmevolumen soll 750 ml (einschließlich antikoagulans, zuzüglich untersuchungsproben) nicht überschreiten. an spender für die granulozytapherese und für die gewinnung allogener blutstammzellen sind besondere anforderungen zu stellen. die spende dieser präparate ist stets eine gerichtete spende für einen bestimmten patienten und unterliegt besonderen vorschriften [15] [66] . granulozytapheresespender müssen mit zytokinen und/oder kortikoiden konditioniert werden. vor beginn der konditionierung soll die leukozytenzahl nicht unter 3 × 10 9 /l und nicht über 13 × 10 9 /l liegen; durch die konditionierung sollen die leukozytenwerte nicht über 70 × 10 9 /l ansteigen. beim einsatz von steroiden sollte eine blutzuckerbestimmung durchgeführt werden. die ärztliche feststellung der spendereignung sollte nicht länger als eine woche vor der apherese liegen. während der apherese gelangt aufgrund des verfahrens (7 abschn. 16.7.2) neben dem antikoagulans auch ein sedimentationsbeschleuniger (in der regel hydroxyethylstärke) in die zirkulation des spenders. eine schwangerschaft muss durch geeignete testverfahren ausgeschlossen sein. ein spender darf nicht mehr als vier granulozytapheresespenden pro jahr leisten. der spendebereich, in dem die entnahme durchgeführt wird, soll abgesondert, ausschließlich für diesen zweck bestimmt und möglichst ruhig sein. neben einer geordneten spendeentnahme ist der schutz der persönlichkeitssphäre des spenders sicherzustellen. die blutentnahme wird durch einen arzt oder unter aufsicht eines arztes von entsprechend ausgebildetem medizinischem assistenzpersonal durchgeführt. die notfallmedizinische versorgung des spenders muss gesichert sein. werden im rahmen der hämapherese geräte eingesetzt, die nach dem prinzip des extrakorporalen kreislaufs arbeiten, müssen diese den vorschriften der medizinprodukte-betreiberverordnung (mpbetreibv) [44] bzw. des medizinproduktgesetzes (mpg) [43] entsprechen. alle eingesetzten materialien, behältnisse und konservierungslösungen müssen pyrogenfrei, steril und gemäß den richtlinien amtlich zugelassen und chargengeprüft sein [18] bzw. den bedingungen der europäischen arzneibuches [16] entsprechen. da die blutentnahme in der regel durch eine andere person als durch den untersucher erfolgt, ist eine identifizierung des spenders unmittelbar vor der blutspende erforderlich. zur gewährleistung der identität des blutes und der für laboruntersuchungen erforderlichen blutproben sind alle für einen spender vorbereiteten behältnisse vor beginn der blutentnahme zu kennzeichnen (name, nummer). unmittelbar vor der blutentnahme und nachdem der spender sich auf die spendeliege gelegt hat, ist er eindeutig positiv zu identifizieren (z. b. durch überprüfung von name und geburtsdatum) und die identität der kennzeichnung an dem blutbehälter und den probenröhrchen nochmals zu überprüfen. nach dem anlegen einer blutdruckmanschette am oberarm (günstiger als eine einfache staubinde, bei welcher der druck nicht kontrolliert werden kann) wird der manschettendruck auf die höhe des diastolischen drucks eingestellt und der spender aufgefordert, die hand zur faust zu schließen. unter den so gestauten kubitalvenen kann die am besten geeignete ausgewählt werden (wenn möglich, zentral gelegen, da bessere lagerung der kanüle). die reinigung der haut im durchmesser von ca. 5 cm um die einstichstelle hat mit einem für die hautdesinfektion anerkannten desinfektionsmittel [41] zu erfolgen, das mit einem sterilen tupfer mehrmals wischend aufgetragen werden soll. im anschluss an die reinigung soll dasselbe desinfektionsmittel erneut aufgetragen werden; nach ablauf der vorgeschriebenen einwirkzeit erfolgt die punktion ohne nochmalige palpation der vene, am besten leicht von der seite, was für den fall, dass der spender durch öffnen und schließen der faust den blutfluss unterstützen muss, die lage der nadel stabilisiert. nach der punktion wird die für untersuchungsproben erforderliche blutmenge zunächst in einen nebenbeutel geleitet; mit dieser maßnahme soll die gefahr der bakteriellen kontamination des vollblutes (durch hautkeime) reduziert werden (sog. »predonation sampling«) [13] . danach wird die sperre am eigentlichen konservenschlauch gelöst; der druck in der manschette sollte knapp unter den diastolischen wert eingestellt werden. für einen ungehinderten blutfluss wird der blutbehälter unterhalb der ebene der einstichstelle platziert. da die füllung der plastikbeutel durch schwerkraft erfolgt, ist eine verlangsamung der fließgeschwindigkeit in der regel nicht erforderlich. bei bedarf kann sie durch veränderung des druckes in der druckmanschette beeinflusst werden. um einer gerinnselbildung im blutbehälter vorzubeugen, ist mehrfaches durchmischen während der blutentnahme unerlässlich. hierfür sind unterschiedliche typen von mischgeräten auf dem markt. ihr gemeinsames funktionsprinzip ist eine andauernde kipp-oder schaukelbewegung des blutbehälters während der blutentnahme. in der regel sind diese geräte mit einer einstellbaren gewichtsmessung (mischwaage) versehen, die das erreichen der gewünschten blutmenge im beutel akustisch oder optisch anzeigt. die gesamtdauer der vollblutspende soll 15 min nicht überschreiten, um das risiko einer gerinnselbildung im schlauchsystem, das frei von antikoagulanzien ist, zu vermeiden. auf die einhaltung des vorgeschriebenen mischverhältnisses zwischen blut und konservierungslösung ist zu achten; in der regel verschließt eine elektronische klemme an der waage den zufluss, wenn das sollgewicht erreicht wurde. wenn der blutbehälter gefüllt ist, wird der entnahmeschlauch mit der sperre verschlossen, der staudruck aufgehoben, die kanüle aus der vene gezogen und die einstichstelle mit einem tupfer bedeckt. der spender soll bei gestrecktem, erhobenem arm (nicht im ellbogen knicken, da so leichter hämatome entstehen) den tupfer gegen die einstichstelle drücken, die in der regel nach 3-5 min verschlossen ist und nun verbunden werden kann. der obere schlauchanteil mitsamt der kanüle kann durch schweißung von der vollblutkonserve getrennt werden. während und mindestens 10 min nach der blutspende darf der blutspender nicht unbeaufsichtigt bleiben. zur vorbeugung von schwindel sind plötzliche veränderungen der körperlage (aufsetzen, aufrichten) zu vermeiden. nach möglichkeit sollten dem spender nach der blutspende ein kleiner imbiss und ausreichend getränke gereicht werden. der spender muss darauf hingewiesen werden, dass er frühestens 30 min nach der spende am öffentlichen straßenverkehr teilnehmen kann. für bestimmte betätigungen (z. b. personenbeförderung) können längere wartezeiten erforderlich sein. bei etwa 2-5 % der blutspender kommt es nach einem initialen anstieg von blutdruck und herzfrequenz zu einer paradoxen, parasympathikotonen reaktion mit vasodilatation, bradykardie und hypotension (vasovagale reaktion) [49] . die neigung zu diesem reaktionstyp ist umso ausgeprägter, je jünger der spender ist, je niedriger das körpergewicht und je kleiner die anzahl der zuvor geleisteten spenden sind [78] ein großteil der vasovagalen reaktionen tritt im nachsorgebereich auf, sodass rund 10 % der spender mit vasovagalen reaktionen folgeverletzungen durch sturzereignisse davontragen, insbesondere schürfwunden [50] . der für den spendebereich verantwortliche arzt muss im einzelfall entscheiden, ob eine weitere diagnostik und/oder therapie erforderlich ist. da für blutspender in deutschland eine allgemeine unfall-und wegeversicherung besteht, sollte in verletzungsfällen die vorstellung beim durchgangsarzt nicht versäumt werden. zur vermeidung derartiger zwischenfälle während und nach der blutspende sollte der blutspender ausgeruht und entspannt zur blutspende erscheinen, nicht unter zeitdruck stehen und nach der spende genügend flüssigkeit zu sich nehmen. es sollte darauf geachtet werden, dass die kleidung nicht zu beengt ist und dass der spender nach der spende nicht sofort aufsteht. die möglichkeit zur körperlichen und psychischen entspannung sollte gegeben sein. der spender soll die spendeeinrichtung erst dann verlassen, wenn er sich vollkommen beschwerdefrei fühlt. z seltene zwischenfälle hämatome sind gelegentlich zu beobachten und in aller regel unbedenklich, können durch schwellung und schmerz den blutspender aber verunsichern. kommt es während der spende zur ausbildung eines größeren hämatoms, sollte diese abgebrochen werden. ein straffer, aber nicht zu fester kompressionsverband sollte 1 h angelegt bleiben. der spender sollte den arm für etwa 6 h nach der punktion nicht übermäßig beanspruchen (z. b. nicht schwer heben). überempfindlichkeitsreaktionen auf desinfektionslösungen oder verbandsmaterialien sind gelegentlich beobachtet worden. sehr seltene ereignisse, die im zusammenhang mit blutspenden berichtet wurden, sind arterielle pseudoaneurysmen und arteriovenöse fisteln nach fehlpunktion einer arterie, verletzungen von ästen des n. medianus und n. ulnaris, lokale wundinfektionen und/oder thrombophlebitiden und kompartmentsyndrome des arms durch einblutungen in die muskellogen. über eintreten und dauer eines jeden zwischenfalls ist ein protokoll zu führen, das den spenderakten hinzugefügt wird. der zwischenfall ist vom arzt nachträglich mit dem spender zu besprechen. während und nach häm-und plasmapheresen mit zellseparatoren können zwischenfälle besonderer art auftreten. nebenwirkungen durch citrat, die sich insbesondere durch parästhesien und metallischen geschmack auf der zunge manifestieren, sind häufiger zu beobachten und können in der regel durch die orale gabe von calcium unterbunden werden. schwere formen jedoch, die die unterbrechung der apherese oder die i.v.-gabe von calcium erfordern, sind selten (ca. 0,5 % aller apheresen) [42] . bereits im ersten weltkrieg setzte o. h. robertson glasflaschen mit citrat-glucose-lösung ein. glasflaschen blieben bis in die 1970er jahre im gebrauch, anschließend wurden sie komplett von kunststoffbeuteln abgelöst. um das risiko der übertragung von syphiliserregern zu reduzieren, wurde eine schnelle abkühlung und lagerung von vollblut bei 1-6 °c mindestens 72 h vor transfusion angestrebt. die transfusion von vollblut wurde gängige praxis. unter den vollblut-lagerungsbedingungen kam es jedoch zu einem erheblichen verlust an funktionstüchtigen gerinnungsfaktoren. daraus wiederum ergab sich die notwendigkeit zu einer möglichst frühen trennung von zellen und plasma, damit letzteres zur bewahrung der gerinnungsaktivität rasch eingefroren werden konnte. die glasflasche ermöglichte diesen ersten schritt in richtung blutkomponentenseparation, da sie zentrifugiert und das plasma vom zellsediment getrennt werden konnte. das so gewonnene plasma konnte transfundiert oder als ausgangsmaterial zur gewinnung von gerinnungsfaktoren, albumin, immunglobulinen und anderen plasmabestandteilen verwandt werden. die trennung von vollblut in ein erythrozytenkonzentrat (mit thrombozyten und leukozyten) und gefrierplasma hatte darüber hinaus auch den vorteil der volumenreduktion. da es sich bei dieser form der blutkomponentenherstellung aber um ein verfahren im offenen system handelte, bestand das erhöhte risiko bakterieller kontamination, ganz abgesehen vom glasbruchrisiko während der zentrifugation. einen wesentlichen fortschritt hinsichtlich der handhabung und zur reduktion des kontaminationsrisikos bei der blutkomponentenherstellung stellte die in den 1950er jahren vollzogene einführung von kunststoffbeuteln dar. um die durch zentrifugation getrennten blutkomponenten ohne kontaminationsgefahr voneinander trennen zu können, wurden durch schläuche miteinander verbundene zweifach-, später auch dreifach-bzw. vierfachbeutelsysteme (. abb. 16 .1) für gefrierplasma, erythrozyten-und thrombozytenkonzentrat sowie den »buffy coat« entwickelt. mehrfachbeutelsysteme ermöglichen heute zudem die leukozytenreduktion durch filtration im geschlossenen system (sog. »inline-filtration«). mit einführung der gezielten thrombozytensubstitution anfang der 1960er jahre in der behandlung von leukämiepatienten und dem damit einhergehenden erhöhten thrombozytenbedarf begann die ära der gewinnung von thrombozyten aus der vollblutspende und durch maschinelle zellseparation (hämapherese). die fraktionierung von vollblut in erythrozytenkonzentrat, gefrierplasma und ggf. thrombozytenkonzentrat ist heute standard. die transfusion von vollblutkonserven wird weitgehend als obsolet angesehen, da wichtige bestandteile wie gerinnungsfaktoren und thrombozyten während der vollblutlagerung bereits nach kurzer zeit funktionell inaktiv werden. walter u. murphy [81] führten 1952 den urtyp des modernen kunststoffbeutels ein. er bestand aus einem kollabierbaren beutel, der mit acd-lösung gefüllt war, sowie einem integrierten schlauch aus pvc (polyvinylchlorid) mit punktionsnadel. die günstigen eigenschaften hinsichtlich verformbarkeit und kollabierbarkeit (von der füllung bei der spende zur entleerung bei der transfusion), temperaturverträglichkeit (+120 °c bei der autoklavierung, -70 °c bei der lagerung von gefrierplasma), widerstandsfähigkeit (bei der zentrifugation) und blutkompatibilität haben dazu beigetragen, dass blutbeutel aus pvc mit zugesetzten weichmachern bis heute zur anwendung kommen. neben dem weichmacher dehp (di-[2ethylhexyl]phthalat) wird auch tehtm (tri-[2-ethlhexyl]trimellitat) sowie in manchen europäischen ländern (spanien, norwegen, schweden) bthc (butyryl-n-trihexyl-citrat) verwendet. die eingesetzten weichmacher gehen während der lagerung aus der beutelfolie in die blutkomponente über. dehp beispielsweise kann im zytosol und der membranfraktion gelagerter erythrozyten nachgewiesen werden. während aber ein stabilisierender effekt von dehp auf die erythrozytenmembranen und geringere hämolyseraten sicher nachgewiesen werden konnten, waren toxische oder kanzerogene wirkungen der weichmacher bisher nicht sicher nachzuweisen. der weichmacher tehtm reichert sich in wesentlich geringerem maße im blutprodukt an. für thrombozytenkonzentrate kommen neben modifizierten pvc-beuteln auch solche aus polyolefin zum einsatz, die frei von weichmachern sind (7 abschn. 16.4.6). acd ist eine mischung aus zitronensäure (acidum citricum), natriumcitrat und dextrose. um das karamelisieren der zur ernährung der erythrozyten zugefügten glucose in lösung während der sterilisation zu verhindern, hatten loutit und mollison zitronensäure zur ansäuerung verwendet. mit überraschung wurde festgestellt, dass dadurch auch die lebensfähigkeit der erythrozyten im blutbeutel deutlich verlängert werden konnte. glucose-citrat-lösungen wurden daraufhin allgemein in die blutkonservierung eingeführt, und es existieren heute viele modifikationen, die sich in ihrer zusammensetzung nur geringfügig unterscheiden. da bei den meisten das verhältnis zwischen zitronensäure und citrat gleich ist, liegt auch der ph-wert der meisten lösungen um 5. nach der mischung mit blut im verhältnis 4:1 (±10 %) ergibt sich durch die proteinpuffer des plasmas ein ph von 7,0-7,1. die einhaltung der vorgeschriebenen mischverhältnisse von stabilisatorlösung und blut ist von wesentlicher bedeutung für die erhaltung der überlebensfähigkeit von erythrozyten. die lagerungszeit der acd-konserve beträgt 21 tage; unzureichende konservenfüllung vermindert die lebensfähigkeit der gelagerten erythrozyten stark [10] . der abfall des ph-wertes in der konserve über die zeit ist eine folge der anaeroben glykolyse durch die erythrozyten mit freisetzung von milchsäure als stoffwechselprodukt. durch den zusatz von natriumphosphat als puffersubstanz in der cpd-lösung kann der ph-wert stabilisiert werden [24] . ein nicht genau eingehaltenes mischverhältnis zwischen stabilisatorlösung und entnommener blutmenge hat in cpd-lösungen einen geringeren einfluss auf die lebensfähigkeit der erythrozyten als in acd-lösungen [9] . von den meisten untersuchern wird der prozentsatz der lebensfähigen, in cpd-lösungen gelagerten erythrozyten etwas höher angegeben im vergleich zu acd-lösungen; es gibt aber auch gleichlautende befunde. [29] . daher ist die zweistufenmethode heute das am häufigsten eingesetzte verfahren zur gewinnung von erythrozytenkonzentraten. leukozyten in blutkomponenten können nach der transfusion beim empfänger zahlreiche unerwünschte wirkungen auslösen. hierzu gehört die febrile, nichthämolytische transfusionsreaktion, die alloimmunisierung gegen hla-antigene, die übertragung leukozytenständiger krankheitserreger (z. b. htlv-i, cmv, ebv, yersinia enterocolitica), die graft-vs.-host-krankheit und die beeinflussung immunologischer funktionen beim empfänger. eine reduktion der transfundierten leukozyten auf unter 5 × 10 7 zellen verhindert die meisten der benannten unerwünschten wirkungen, ausgenommen die graft-vs.-host-krankheit, zu deren verhinderung die blutprodukte bestrahlt werden müssen. die effektive abreicherung von leukozyten wird durch filter erreicht, die aus mehreren schichten nichtgewebter, synthetischer fasern aufgebaut sind (übersicht bei [8] [57] . nach 24 h ist mit vermehrtem zerfall der granulozyten und freisetzung phagozytierter, nicht abgetöteter mikroorganismen zu rechnen, die andernfalls durch filtration entfernt würden. nach den vorgaben der aabb [77] enthält eine leukozytendepletierte einheit (erythrozyten-oder thrombozytenkonzentrat) weniger als 5 × 10 6 leukozyten, nach den empfehlungen des europarates [14] sind es weniger als 1 × 10 6 leukozyten; die verfügbaren filtersysteme ermöglichen die abreicherung auf den europäischen grenzwert. in deutschland dürfen nur noch erythrozyten-und thrombozytenkonzentrate in verkehr gebracht werden, die weniger als 1 × 10 6 leukozyten pro einheit enthalten. nach den für deutschland gültigen richtlinien [66] tel zu einer höheren thrombozytenausbeute. zum teil finden auch längere lagerzeiten des »buffy coat« von 12 h bzw. über nacht mit und ohne bewegung anwendung. die buffy-coat-beutel werden bei niedriger g-zahl zentrifugiert und der plättchenreiche überstand abgepresst. um die leukozytenkontamination möglichst niedrig zu halten, wird empfohlen, 1 cm oberhalb des leuko-und erythrozytensediments den pressvorgang zu beenden [58] . die in deutschland übliche standardpräparation besteht im zusammenführen (»poolen«) von 4-6 abo-blutgruppen-gleichen »buffy coats« mit plasma oder additivlösung für thrombozyten (. tab. 16 .5) in einem »poolingbeutel«. dazu werden die »buffy coats« sorgfältig durchmischt und zusammen mit autologem plasma aus einer der 4-6 spenden oder einer speziellen additivlösung für thrombozytenkonzentrate über ein »poolingset« steril an einen beutel angeschweißt. die »buffy coats« werden in den »poolingbeutel« überführt, und mit dem plasma oder der additivlösung werden die entleerten buffy-coat-beutel ausgespült, um möglichst alle thrombozyten zu gewinnen. anschließend erfolgt die zentrifugation (480 g, 13 min). der plättchenreiche überstand kann dann durch einen leukozytendepletionsfilter in den lagerbeutel überführt werden. das poolen und isolieren der thrombozyten aus den 4-6 »buffy coats« kann auch maschinell erfolgen (orbisac ® , fa. caridian; tacsi ® , fa. terumo). der vorteil dieses verfahrens liegt in der gewinnung eines leukozytendepletierten pool-thrombozytenkonzentrates mit einer standarddosis thrombozyten für erwachsene bei minimaler restleukozytenzahl (<1 × 10 6 ). das produkt wird bei 22±2 °c und unter ständiger agitation gelagert [19] [20] . die herstellung nach dem plättchenreichen-plasma-(prp-)verfahren erfolgt in der regel im dreifachbeutelsystem. nur wenn die leukozytenhaltige plasma-zell-grenzschicht des erythrozytenkonzentrats entfernt werden soll, benötigt man ein vierfachbeutelsystem. das vollblut wird zuerst langsam zentrifugiert. kurze zentrifugationszeiten mit entsprechend erhöhter g-zahl sollen die lagerfähigkeit der thrombozyten verbessern [74] . das plättchenreiche plasma wird in den thrombozytenbeutel abgepresst und anschlie-ßend durch eine hochtourige zentrifugation in thrombozyten (sediment) und plättchenarmes plasma getrennt. die sedimentierten thrombozyten werden nach 1-2 h vorsichtig resuspendiert. nachteilig beim prp-verfahren ist die thrombozytenaktivierung (verlust der diskoiden form, plättchenfaktor-4-und β-thromboglobulinfreisetzung, erhöhte expression von gpiib/iiia und cd62p) [23] [82] . dies wird auf die pelletierung an die beutelwand bei der zweiten, hochtourigen zentrifugation beim prp-verfahren zurückgeführt. plättchenreiches plasma wird heute wegen der volumenbelastung (6-8 × 10 10 erwägungen, dass erreger, auf die blutspenden (noch) nicht getestet werden, die empfängersicherheit beeinträchtigen könnten, haben der entwicklung von verfahren vorschub geleistet, deren ziel die inaktivierung von infektionserregern in der blutkomponente ist. die inaktivierungsmechanismen basieren entweder auf der solubilisierung von lipidmembranen durch detergenzien sowie auf der direkten (z. b. uv-c-behandlung) oder indirekten schädigung der erbsubstanz von viren und bakterien. bei letzterer werden den blutkomponenten substanzen zugesetzt, die nach aktivierung durch licht (»photosensitizer«) oder durch ph-verschiebung sich mit der mikrobiellen erbsubstanz verbinden (z. b. vernetzung der dna-stränge) und/oder zur bildung von photoxydanzien führen, welche mit den nukleinsäuren reagieren. diese reaktionen mit der erbsubstanz hemmen deren replikationsfähigkeit. für frischplasma kommen schon lange solvent/detergent-(sd-) verfahren unter einsatz von tri-n-butyl-phosphat und detergenzien wie triton x-100 zum einsatz [35] , welche lipidmembranen zerstören. nach inkubation müssen die substanzen durch öl und adsorptionschromatographie wieder aus dem plasma entfernt. es hat sich gezeigt, dass die sd-behandlung zu einem abfall von fv, fviii, protein s, antitrypsin und antiplasmin führt [71] . aufgrund des wirkprinzips werden nur infektionserreger mit einer lipidhülle inaktiviert, jedoch nicht z. b. nichtumhüllte viren, auch kann das verfahren nicht für zellhaltige blutkomponenten eingesetzt werden. ebenso auf plasma beschränkt bleibt der einsatz des phenothiazinfarbstoffs methylenblau. nach zusatz zum plasma (1 μmol/l) und photodynamischer aktivierung (uv-bestrahlung bei 590 nm) kommt es zur interkalierung in im plasma vorhandener nukleinsäurestränge sowie zur freisetzung von sauerstoffradikalen, die zur irreversiblen schädigung der nucleinsäuren führen [83] . da methylenblau wenig in bakterien, protozoen und restzellen eindringt, werden diese auch unvollständig inaktiviert. nach behandlung werden methylenblau, seine photoderivate sowie vorhandene restzellen durch einen filter aus dem plasma entfernt. es ist bekannt, dass die entstehenden photoxydanzien auch zu einem abfall der fibrinogen-und fviii-aktivität um 20-35 % in den behandelnden plasmen führen [84] . prinzipiell können auch psoralene (amotosalen) und riboflavin (vit. b 2 ) nach zusatz und photodynamischer aktivierung zur pathogeninaktivierung von frischplasma eingesetzt werden, bisher haben sie aber noch keine breitere anwendung gefunden. für thrombozytenkonzentrate ist es wichtig, dass auch intrazelluläre erreger ohne signfikante schädigung der thrombozyten inaktiviert werden. photoaktive moleküle aus der familie der psoralene können als kleine planare moleküle zellmembranen durchdringen. sie interkalieren mit der erbsubstanz. nach bestrahlung mit uv-licht (320-400 nm) entstehen addukte mit den pyrimidinbasen, die zu einer irreversiblen vernetzung der nukleinsäurestränge führen. dieses »cross-linking« behindert die replikationsfähigkeit des krankheitserregers bzw. der zelle. psoralen und nebenprodukte werden durch photodegradation sowie einen absorptionsfilter aus der blutkomponente wieder entfernt [40] . auch riboflavin (vitamin b 2 ) bindet durch interkalation an die dna. uv-licht induziert die oxidation der nucleinsäure guanin. folge sind einzelstrangbrüche und die ausbildung kovalenter addukte, was ebenfalls zur unterbindung der replikationsfähigkeit der krankheitserreger führt [70] . neuere untersuchungen beschäftigen mit der direkten schädigung des erbguts der infektionserreger durch uv-c-belichtung. agitation der thrombozytenkonzentrate während der uv-c-behandlung scheint die antiinfektiöse wirkung wesentlich zu verstärken. bei pathogeninaktivierten thrombozytenkonzentraten ist die recovery-rate ca. 20 % niedriger und das überleben der thrombozyten in vivo 20-27 % kürzer als bei nichtinaktivierten thrombozytenkonzentraten [85] . der einsatz photoaktiver substanzen in erythrozytenkonzentraten wird durch die physikalischen eigenschaften des hämoglobins erschwert. hämoglobin streut und absorbiert uv-und sichtbares licht bis 700 nm. membrangängige substanzen (s-303, pen110), die ohne aktivierung mittels belichtung nukleinsäuren irreversibel schädigen, wurden erprobt. wegen ihrer potenziellen mutagenität müssen diese substanzen nach behandlung wieder aus der blutkomponente entfernt werden. da es in der vergangenheit zur induktion von antikörpern bei den transfusionsempfängern kam, wurden die klinischen studien zunächst ausgesetzt [84] . es bleibt abzuwarten, ob durch modifizierte verfahren die bildung von erythrozytären antikörpern verhindert werden kann. die pathogeninaktivierungsspektren aller verfahren sind nicht umfassend. die behandelnden präparate weisen spezifische beeinträchtigungen auf. beim einsatz von pathogeninaktivierungsverfahren ist (noch) mit erheblichen mehrkosten bei der herstellung von blutkomponenten zu rechnen. schließlich wird das sicherheitsprofil (risiko der induktion von autoimmunerkrankungen oder tumoren) der zur zeit eingesetzten photoaktiven substanzen kontrovers beurteilt, da langzeituntersuchungen fehlen. ob diese nachteile durch den vermuteten nutzen aufgewogen werden, ist gegenstand der diskussion [84] . optimale lagerungsbedingungen sind voraussetzung für die erhaltung der funktionsfähigkeit der einzelnen blutbestandteile. daher wird das vollblut in einem definierten zeitfenster fraktioniert und die gewonnenen blutbestandteile entsprechend gelagert: frischplasma, um einem verlust an gerinnungsaktivität zu begegnen, tiefgefroren; thrombozyten, um einen funktionsverlust zu vermeiden, bei raumtemperatur; und erythrozyten bei 4 °c, damit ein reduzierter stoffwechsel möglichst lange lagerzeiten erlaubt und zugleich das bakterielle wachstum gehemmt wird. einzelheiten der kryokonservierung von blut-und knochenmarkzellen werden in 7 kap. 10 behandelt. erythrozyten werden nach der gewinnung aus vollblut in eine additive lösung überführt, die die voraussetzungen für möglichst lange lebensfähigkeit unter lagerungsbedingungen bietet. bei dem cpd/sag-m-system wird vollblut in einen cpd-beutel entnommen, und nach der zentrifugation werden die vom plasma getrennten erythrozyten in der sag-m-lösung (sodium, adenin, glucose, mannitol) auf einen hämatokritwert von etwa 60-70 % aufgeschwemmt und bei 4 °c gelagert. nach 42 tagen lagerungszeit beträgt die 24-h-überlebenszeit der erythrozyten in vivo 73,3 %. nach 4 wochen lagerungszeit ist der atp-gehalt der erythrozyten nahezu normal, morphologische veränderungen und hämolyse sind mäßig (weniger als 0,8 % der erythrozytenmasse am ende der lagerungszeit). mit der konservierungslösung paggs-sorbit (saure phosphate, adenin, guanosin, glucose, sorbit) wurden günstige hämolysetrotz verbesserter konservierungs-und lagerungsbedingungen sind veränderungen des erythrozytenstoffwechsels während der lagerung nicht zu vermeiden (lagerungsschäden). schon in den 1950er jahren wurde festgestellt, dass sich die o 2 -dissoziationskurve in gelagerten blutkonserven bereits nach einer woche nach links verschiebt, was bedeutet, dass diese erythrozyten im gewebe nicht dieselbe menge sauerstoff freisetzen können wie frisch entnommene. nach transfusion normalisiert sich diese linksverschiebung im verlauf von 24 h. die linksverschiebung der dissoziationskurve geht mit dem verlust von 2,3-dpg einher. auch die im cpd-blut im vergleich zum acd-blut geringer ausgeprägte linksverschiebung der dissoziationskurve wird auf einen höheren 2,3-dpg-spiegel zurückgeführt. die klinische bedeutung des im konservenblut verminderten 2,3-dpg-spiegels ist noch immer umstritten. es wird angenommen, dass dies nur unter kritischen bedingungen (z. b. begrenzte myokardreserven, koronarinsuffizienz) zum tragen kommen kann. der atp-gehalt der erythrozyten vermindert sich im laufe der lagerung zunehmend. parallel dazu stellen sich ein lipidverlust der zellmembranen, sphärozytose und ein anstieg der rigidität der zellen ein [11] . wurde so veränderten erythrozyten adenin und inosin zugesetzt, so gewannen sie ihre ursprüngliche diskoide form wieder, ihr atp-gehalt näherte sich dem normalwert und die 24-h-überlebenszeit in vivo betrug 90 %. kommt es zu einer atp-verminderung, so treten die formveränderungen vor der abnahme der verformbarkeit auf [22] . die während der lagerung in konservierungslösungen gesteigerte schwellung und hämolyse der erythrozyten wird durch zugabe von mannitol verhindert [6] . veränderungen der verformbarkeit von erythrozyten zeigen dabei eine relativ gute korrelation mit ihrer überlebenszeit in vivo [86] . die normale lebensfähigkeit der erythrozyten nach der übertragung in den empfängerorganismus ist der wichtigste parameter, an welchem der erfolg der lagerung von blut und blutbestandteilen gemessen werden kann. verlässliche angaben über die lebensfähigkeit können nur mit in-vivo-methoden gewonnen werden, wobei in der regel isotopenmethoden, z. b. unter verwendung von 51 chrom, durchgeführt werden. alle in der konserve vorhandenen, bereits lädierten oder abgestorbenen erythrozyten werden in den ersten 24 h nach der transfusion im organismus abgebaut. erythrozyten, die diese zeitspanne überlebt haben, altern normal, soweit dies nicht durch extra-erythrozytäre umstände (z. b. antikörper) negativ beeinflusst wird. als maß für den wert der erythrozytenkonservierung wird der prozentsatz der erythrozyten angegeben, der länger als 24 h im empfängerkreislauf überlebt. dieses kriterium sollen mindestens 70 % der transfundierten zellen erfüllen. vor der einführung der allgemeinen leukozytendepletion war die bildung von mikroaggregaten in erythrozytenkonzentraten gegenstand zahlreicher untersuchungen. insbesondere im rahmen der massivtransfusion maß man ihnen klinische bedeutung bei und verwendete mikrofilter bei der transfusion. mit einführung der leukozytendepletion kann davon ausgegangen werden, dass sich mikroaggregate in erythrozytenkonzentraten nicht mehr in nennenswertem umfang bilden können. damit ist auch der einsatz von mikrofiltern heute obsolet. eine erhöhte aggregationsbereitschaft der erythrozyten (sog. rouleau-bildung) in abhängigkeit von der lagerungszeit ist in vitro nachweisbar [30] . während die lagerungszeit keinen einfluss auf die blutgruppenmerkmale abo und rh hat, wurde mit fortscheitendem alter der konserve ein reaktivitätsverlust der blutgruppenmerkmale lewis und p beschrieben [48] . zuckerreste auf der erythrozytenoberfläche, die im verlauf der lagerung exprimiert werden, können antigenen charakter haben und beispielsweise eine positive serologische verträglichkeitsprobe hervorrufen [37] . nach den richtlinien soll frischplasma möglichst innerhalb 6-8 h nach der spende, spätestens jedoch nach 24 h eingefroren werden. zwar bleiben nahezu alle gerinnungsfaktoren über diesen zeitraum stabil, doch faktor viii zeigt bereits nach 8 h lagerung bei raumtemperatur eine abnahme um 13 % und nach 24 h um weitere 15-20 % [51] . die lagerung des plasmas erfolgt bei -30 bis -40 °c (±3 °c) über 1 jahr oder die ermittelte haltbarkeit. die mindestlagerzeit beträgt 4 monate, da nach den bestimmungen der richtlinien frischplasma erst dann therapeutisch eingesetzt werden darf, wenn bei einer nachfolgenden spende oder blutentnahme keine infektiologischen auffälligkeiten vorlagen (quarantäneplasma). von dieser bestimmung ausgenommen ist pathogeninaktiviertes plasma, das als gepooltes produkt einer virusinaktivierung unterzogen wurde. die vorgaben der aabb [77] sehen eine 8-h-frist zur tiefkühlung bei -18 °c für plasma aus cpd-oder cpda-1-vollblut vor und eine 6-h-frist für acd-plasma, das durch apherese gewonnen wurde. bei einer lagerungstemperatur von -18 °c oder darunter wird die lagerzeit mit 1 jahr angegeben. nach dem auftauen in zugelassenen wärmegeräten bei maximal 37 °c oder in für diesen zweck zugelassenen mikrowellengeräten ist das frischplasma zur unmittelbaren anwendung bestimmt [66] . die bestimmungen der aabb [77] hingegen erlauben eine weitere lagerung bei 1-6 °c über maximal 24 h; erst nach diesem zeitraum ist von einer klinisch signifikanten reduktion des gehaltes an faktor viii auszugehen. im hinblick auf ihre hämostatische funktionsfähigkeit können thrombozyten in speziellen, gasdurchlässigen beuteln bei raumtemperatur (20-24 °c) und unter ständiger agitation bzw. rotation mehrere tage gelagert werden. die bemühungen, die lagerungszeit von thrombozytenkonzentraten auf bis zu 7 tage zu verlängern, werden durch die möglichkeit von bakteriellen kontaminationen eingeschränkt, die zu schweren septischen zwischenfällen geführt haben (7 kap. 21, 35 [80] . eine 6-bis 12-stündige unterbrechung der agitierten lagerung zu transportzwecken mit darauf folgender erneuter lagerung im rotator bis zu einer gesamtlagerungszeit von 72 h soll zu keiner verminderung der recovery-rate und der in-vivo-überlebenszeit führen [73] . granulozytenkonzentrate sind zur umgehenden transfusion bestimmt und sollten innerhalb von 6 h transfundiert werden. die richtlinien der aabb [77] in den usa sehen für granulozytenkonzentrate eine maximale lagerzeit von 24 h bei 20-24 °c ohne bewegung vor. die bestrahlung von blutkomponenten mit ionisierenden strahlen dient der verhinderung des anwachsens transfundierter lymphozyten im empfänger und damit der ausbildung einer graft-vs.-host-reaktion (gvhr). bereits 10 4 transfundierte zellen pro kg körpergewicht des patienten werden als ausreichend zur auslösung einer gvhr angesehen [38] . durch die bestrahlung wird die dns der lymphozyten irreversibel geschädigt. empfänger bestrahlter blutpräparate sind in erster linie stark immungeschwächte patienten (wie knochenmark-/blutstammzell-transplantierte, patienten vor autologer blutstammzellentnahme, patienten mit angeborenen immundefekten sowie feten im rahmen der intrauterinen transfusionen); ebenfalls bestrahlt werden müssen blutkomponenten, die von angehörigen ersten grades stammen, etwa bei transfusion von elterlichem blut (»one way hla-mismatch«). eine übersicht über die indikationen geben die leitlinien der bundesärztekammer [62] . granulozytenkonzentrate sind aufgrund ihres herstellungsbedingt hohen gehaltes an lymphozyten immer zu bestrahlen. die notwendige energiedosis zur vermeidung einer gvhr liegt nach experimentellen untersuchungen in der größenordnung von 30 gy [52] [69] . die richtlinien empfehlen, blutpräparate mit einer mittleren dosis von 30 gy zu bestrahlen, wobei die energiedosis an keiner stelle des präparates 25 gy unterschreiten darf. die bestrahlung der blutkomponenten erfolgt in eigens für diese anwendung hergestellten bestrahlungsgeräten [46] . die blutpräparate werden dabei in einen zylinderförmigen behälter einer drehbaren bleitrommel eingelegt. durch rotation der trommel um 180° wird die blutkomponente in die nähe der bestrahlungsquelle gebracht. in der regel kommt 137 caesium, selten 60 cobalt als radioisotop zur anwendung. (prinzipiell wäre die anwendung von röntgenstrahlen, wie sie z. b. in linearbeschleunigern erzeugt werden, dem einsatz von γ-strahlen gleichwertig. sie ist jedoch in der regel technisch ungleich aufwendiger.) um eine möglichst homogene dosisverteilung zu erreichen, dreht sich in den blutbestrahlungsgeräten der zylinder mit dem blutpräparat in der ruhenden trommel kontinuierlich während der gesamten bestrahlungszeit. nach ablauf der bestrahlungszeit rotiert die bleitrommel wieder zur ausgangsposition zurück, und das bestrahlte präparat kann entnommen werden. kontrollstreifen (sog. bestrahlungsindikatoren), die sich oberhalb einer energiedosis von 25 thrombozytapheresekonzentrate sollten 2-4 × 10 11 thrombozyten und höchstens 3 × 10 9 erythrozyten in höchstens 300 ml plasma enthalten. der ph muss zwischen 6,5 und 7,4 liegen. in den leukozytendepletierten präparaten sind weniger als 1 × 10 6 restleukozyten/einheit vorhanden. bei den meisten apheresesystemen ermöglichen die kammereigenschaften eine ausreichende leukozytenabreicherung, sodass diese thrombozytapheresekonzentrate herstellungsbedingt leukozytenarm sind. bei verwendung anderer systeme muss das präparat nach der gewinnung noch gefiltert werden. um während der zentrifugation eine ausreichende trennung von granulozyten und erythrozyten zu erreichen, hat sich der einsatz von hochmolekularer hydroxyethylstärke (hes) als sedimentationsbeschleuniger bewährt. da unverträglichkeitsreaktionen auf hes beschrieben sind, wird eine sog. biologische vorprobe durch intravenöse injektion von 1 ml 6 %iger hes mit anschließender 5-minütiger beobachtungszeit empfohlen [66] . während der apherese werden dem spenderblut dann 200 bis maximal 750 ml 6 %ige hes zugesetzt. granulozytenkonzentrate sollten mehr als 1 × 10 10 granulozyten/m 2 körperoberfläche des empfängers in maximal 500 ml konzentratvolumen enthalten. der hämatokritwert des präparates sollte 30 % nicht übersteigen. granulozytenkonzentrate sind aufgrund des hohen lymphozytenanteils vor der transfusion mit 30 gy zu bestrahlen. die plasmagewinnung kann durch die beschriebenen zellseparatoren erfolgen oder durch geräte, die plasma mittels mechanischer filtration gewinnen. um extrem zellarmes plasma zu erhalten, werden zentrifugation und filtration auch kombiniert angewendet. bei der maschinellen plasmapherese dürfen nicht mehr als 850 ml plasma (mit antikoagulans gerechnet) je spende gewonnen werden. das durch maschinelle plasmapherese gewonnene plasma muss die gleichen qualitätskriterien erfüllen wie das durch konventionelle vollblutspende gewonnene. viele unreife neugeborene, v. a. solche mit einem körpergewicht von weniger als 1 kg, müssen in den ersten lebenswochen regelmäßig transfundiert werden. lange zeit wurde frischen erythrozytenkonzentraten mit einer lagerzeit <7 tage der vorzug gegeben, da mit zunehmender lagerzeit die konzentration an freiem kalium in der additivlösung ansteigt und der gehalt der erythrozyten an 2,3-dpg abnimmt. ein 42 tage altes erythrozytenkonzentrat enthält etwa 50 mmol freies kalium/l extrazelluläre flüssigkeit; bei der üblichen transfusionsmenge von 15±5 ml/kgkg ist die kaliumzufuhr mit 0,1-0,15 mmol/kgkg im vergleich zum tagesbedarf (ca. 2-3 mmol/kgkg) jedoch gering. die verminderung an 2,3-dpg führt zu einem abfall des zur 50 %igen sättigung erforderlichen sauerstoffpartialdruckes (p 50 ) von 27 mmhg (frischblut) auf 18 mmhg. dieser wert entspricht aber den physiologischen verhältnissen, wie sie erythrozyten von frühgeborenen zeigen, allerdings mit dem unterschied, dass der 2,3-dpg-spiegel in den spendererythrozyten nach der transfusion rasch ansteigt und nach wenigen stunden wieder normalwerte erreicht hat. mehrere studien haben mittlerweile gezeigt, dass der transfusionserfolg und die rate unerwünschter wirkungen nicht vom alter des erythrozytenkonzentrates abhängig sind (übersicht bei [76] ). auch die art der verwendeten additivlösung und ihr gehalt an glucose, phosphat und mannitol sind ohne klinische bedeutung, sodass es unnötig ist, sie vor der transfusion kleiner volumina zu entfernen. von austauschtransfusionen abgesehen, ist es unklar, ob bei größeren transfusionsvolumina die additivlösung entfernt werden soll. für intrauterine transfusionen muss die additivlösung weitgehend entfernt werden, damit der empfohlene hämatokritwert von 75-85 % eingestellt werden kann (hämatokrit von erythrozytenkonzentraten in additivlösung ca. 50-70 %); für austauschtransfusionen wird ein hämatokrit von 40-50 % empfohlen, der nach entfernen der additivlösung durch zusatz von plasma eingestellt werden kann [77] . die für die transfusion früh-und neugeborener benötigten kleinen mengen werden in der regel durch verwendung von beutelsystemen mit kleinen satellitenbeuteln (»babybeutel«) bereitgestellt, aus denen spritzen zur transfusion gefüllt werden können. viele neonatologen befürworten eine möglichst geringe spenderexposition, die dadurch erreicht werden kann, dass die satellitenbeutel aus einem konzentrat einem empfänger zugeordnet werden und über die lagerzeit für diesen reserviert bleiben. bei intrauterinen transfusionen und austauschtransfusionen bei neugeborenen ist zu beachten, dass die präparate bestrahlt werden müssen [66] . während rund 80 % der frühgeborenen mit erythrozytenkonzentraten versorgt werden müssen, sind nur 15-20 % auf die transfusion weiterer blutkomponenten angewiesen. für thrombozytensubstitutionen (mittlere dosis: 5-10 ml/ kgkg) kann es nötig werden, das plasmavolumen eines thrombozytenkonzentrates auf 10-20 ml zu reduzieren. in volumenreduzierten thrombozytenkonzentraten, die in spritzen umgefüllt wurden, fällt der ph rasch ab. dieser schritt sollte daher frühestens 4 h vor transfusion durchgeführt werden [59] . da die transfusion inkompatiblen plasmas bei kleinkindern größere gefahren birgt als im höheren lebensalter, soll abo-ident transfundiert werden. müssen thrombozytenkonzentrate zur anwendung kommen, die inkompatibles plasma enthalten, sollen das plasma entfernt und die thrombozyten in kochsalzlösung, albumin oder kompatiblem plasma resuspendiert werden [77] . die übliche dosis für gefrorenes frischplasma ist 10-15 ml/ kgkg. frischplasmen stehen in der regel nicht in kleinen abpackungen zur verfügung und sind nach dem auftauen zur unmittelbaren anwendung bestimmt [66] . mit einem klinisch signifikanten verlust an gerinnungsaktivität (insbesondere faktor viii) ist nach 24 h zu rechnen [77] . zur überführung von blutkomponenten aus beutelsystemen in zusätzliche, nicht bereits angeschlossene beutel muss das eröffnen des kontaminationssicheren »geschlossenen« systems vermieden werden. dies kann erreicht werden, indem mit schweißgeräten (»sterile connecting devices«, stcd) sterile verbindungen zwischen 2 schläuchen konventioneller beutelsysteme hergestellt werden. dazu werden die schläuche in schlauchhalterungen eingelegt, mittels einer auf 320 °c erhitzten kupferklinge durchtrennt, entlang der klinge zur gewünschten verbindung verschoben und während der klingenentfernung unter zusammenführung der schnittenden miteinander steril verschweißt. während des schweißvorganges wird evtl. vorhandene flüssigkeit durch zusammendrücken des schlauches von der schweißnaht getrennt. die flüssigkeit in den schläuchen kann auch durch eine entsprechende schlauchführung (»bend-tube-methode«) von der schweißstelle ferngehalten werden. integrierte temperatursensoren überwachen kontinuierlich die klingentemperatur. die kupferklinge ist nur einmal verwendbar. die zu verbindenden schläuche sollten den gleichen innendurchmesser (in der regel 2,9-3,1 mm) haben und aus demselben material bestehen. verbindungen zwischen zwei trockenen schläuchen weisen 75 % der ziehfestigkeit der ausgangsschläuche auf [36] . der transport von blutkomponenten vom hersteller zum depot des anwenders erfolgt unter der verantwortung des herstellers [66] . von besonderer bedeutung ist die einhaltung der vorgesehenen transporttemperaturen, die während des transportes durch geeignete maßnahmen (z. b. temperaturschreiber oder min/max-thermometer) zu überwachen sind. dabei gilt, dass erythrozytenkonzentrate ohne unterbrechnung der kühlkette bei 1-10 °c transportiert werden sollen. für thrombozytenkonzentrate gilt raumtemperatur als geeignete lagertemperatur, wobei 20 °c nicht unterschritten werden dürfen. gefrierplasma ist tiefgefroren zu transportieren. andere schädigende einflüsse (massive erschütterung u. ä.) sind zu vermeiden. werden blutkomponenten von einem blutdepot zu einem anderen weitergegeben, so geht die verantwortung für deren qualität und unbedenklichkeit sowie die transportverantwortung vom hersteller auf den leiter der weitergebenden einrichtung über. innerhalb der einrichtung des anwenders sollen blutkomponenten nur zur unmittelbaren anwendung am patienten aus dem blutdepot abgerufen werden. der transport soll durch einen eingewiesenen botendienst (und nicht durch besucher, patienten oder deren angehörige) und unter geregelten bedingungen erfolgen. dabei sollten v. a. die art der transportbehältnisse und die transportzeiten geregelt werden. in großen krankenhäusern kann es erforderlich werden, satellitendepots, insbesondere für erythrozytenkonzentrate, einzurichten. für die lagerung in solchen depots gelten dieselben vorschriften wie für die lagerung im blutdepot selbst; satellitendepots sind sorgfältig zu überwachen. eine rücknahme von erythrozytenkonzentraten aus solchen depots ist nur unter definierten bedingungen möglich, wobei neben der sicheren dokumentation der lagertemperatur zumindest die haltbarkeit, die unversehrtheit des beutels und die hämolyse geprüft werden sollten. influence of 30 gy gamma irradition on the quality of red blood cell concentrates in several storage media volume control of erythrocytes during storage. the role of mannitol acute coagulopathy of trauma:mechanism, identification and effect the mechanism of leukocyte removal by filtration effects of white cell reduction on the resistance of blood components to bacterial multiplication the quality of overand undercollected blood for transfusion deformability of stored red blood cells. relationship to degree of packing the selection of plastic materials for blood bags reduction of bacterial load by predonation sampling guide to the preparation, use and quality assurance of blood components, 14 edn durchführung präparativer hämapheresen zur gewinnung von blutbestandteilkonzentraten -empfehlungen zur präparativen hämapherese der deutschen gesellschaft für transfusionsmedizin und immunhämatologie (dgti) eur. 2008) mit nachtrag, amtliche deutsche ausgabe in vivo and in vitro comparison of platelets stored in either synthetic media or plasma platelet concentrates in an additive solution prepared from pooled buffy coats. 1. in vitro studies platelet concentrates in an additive solution prepared from pooled buffy coats. in vivo studies histopathological studies on kidneys from patients treated with largeamounts of blood preserved with acd-adenine clarification of role of atp in red-cell morphology and function platelet activation during preparation of platelet concentrates:a comparison of the platelet-rich plasma and the buffy coat methods a citrate-phosphate-dextrose solution for preservation of human blood effectiveness of white cell reduction by filtration with respect to blood storage time studies in red blood cell preservation. 2. comparsion of vesicle formation, morphology, an membrane lipids during storage in as-1 and cpda-1 additive solutions for the storage of platelets for transfusion preservation of red blood cells:content of microaggregates and di-2-ethylhexylphthalate (dehp) in red blood cells stored in saline-adenine-glukosemanitol (sagm) medium use of adsol preservation solution for prolonged storage of low viscosity as-1 red bood cells cell surface alterations during blood-storage characterized by artificial aggregation of washed red blood cells damage control resuscitation:directly addressing the early coagulopathy of trauma coagulase-negative staphylococcal contamination of whole blood and its components: the effects of wbc reduction white cells protect donor blood against bacterial contamination evaluation and comparison of three mobilization methods for the collection of granulocytes current status of solvent/detergent-treated frozen plasma the use of the sterile connection device in transfusion medicine development of a carbohydrate antigen during storage of red cells dose, dosimetry, and quality improvement of irradiated blood components a comparative trial of granulocyte-colony-stimulating factor and dexamethasone, separately and in combination, for the mobilization of neutrophils in the peripheral blood of normal volunteers inactivation of viruses in platelet concentrates by photochemical treatment with amotosalen and long-wavelength ultraviolet light bdh auf der basis der standardmethoden der dghm zur prüfung chemischer desinfektionsverfahren geprüften und als wirksam befundenen verfahren für die prophylaktische desinfektion und die hygienische händewaschung national audit of citrate toxicity in plateletpheresis donors bundesgesetzblatt teil i bundesgesetzblatt teil i:1762 (1998), in der jeweils gültigen fassung the introduction of citrate as an anticoagulant for transfusion and of glucose as a red cell preservative principles of blood irradiation, dose validation, and quality control viability and in vitro properties of as-1 red cells after gamma irradiation preservation of red cell antigens during storage of blood with different anticoagulant donor reactions and injuries from whole blood donation a study of 178 consecutive vasovagal syncopal reactions from the perspective of safety kapitel 16 • gewinnung, herstellung und lagerung von blut und blutkomponenten 243 effect of 24-hour whole-blood storage on plasma clotting factors effect of gamma-irradiation of red blood cell units on t-cell inactivation as assessed by limiting dilution analysis: implications for preventing transfusion-associated graft vs.-host disease quality assurance and quality control in component preparation platelet concentrates stored in plasma for 72 hours at 22 °c prepared from buffy coats in citrate-phosphate-dextrose blood collected in a quadruple-bag saline-adenine-glukose-mannitol system storage of whole blood for up to 24 h at ambient temperature prior to component preparation preparation of leukocyte-poor platelet concentrates from buffy coats. i. special inserts for centrifuge cups prevention of yersinia enterocolitica growth in red-blood-cell concentrates prestorage leukocyte depletion of blood products in a closed system in vitro characteristics of white cell-reduced single-unit platelet concentrates stored in syringes durchführung apparativer plasmapheresen zur gewinnung von spenderplasma. empfehlungen der ständigen hämapheresekommission der deutschen gesellschaft für transfusionsmedizin und immunhämatologie e prevention of microaggregate formation by removal of buffy-coats bundesärztekammer auf empfehlung ihres wissenschaftlichen beirats high potassium levels in stored irradiated blood viability of platelets following storage in the irradiated stage richtlinien für die herstellung von plasma für besondere zwecke (hyperimmunplasma zweite richtlinienanpassung 2010 the new generation of platelet additive solution for storage at 22 °c:development and current experience the effects of irradiation on platelet function prevention of transfusion-associated graft-vs.-host disease:selection of an adequate dose of gamma radiation photochemical inactivation of selected viruses and bacteria in platelet concentrates using riboflavin and light are quality differences responsible for different adverse reactions reported for sd-plasma from usa and europe red cell preservation: further studies with adenine lack of adverse effect of transportation on room temperature stored platelet concentrates effect of centrifugation on the storage properties of platelets proteomic characterization of freeze-dried human plasma:providing treatment of bleeding disorders without the need for a cold chain data-driven blood banking practices for neonatal rbc transfusions american association of blood banks a case-controlled multicenter study of vasovagal reactions in blood donors: influence of sex, age, donation status, weight, blood pressure and pulse six filters for the removal of white cells from red cell concentrates, evaluated at 4 °c and/or at room temperature wbc-reduced platelet concentrates from pooled buffy coats in additive solution:an evaluation of in vitro and in vivo measures a closed gravity technique for the preservation of whole blood in acd solution utilizing plastic equipment prevention of acquired defects in platelet function during blood processing virus inactivation in blood components by photoactive phenothiazine dyes methylene blue-treated fresh-frozen plasma: what is its contribution to blood safety proceedings of a consensus conference:pathogen inactivation -making decisions about new technologies the membrane and the lesion of storage in preserved red cells key: cord-319013-oytqcifa authors: focosi, daniele; anderson, arthur o.; tang, julian w.; tuccori, marco title: convalescent plasma therapy for covid-19: state of the art date: 2020-08-12 journal: clin microbiol rev doi: 10.1128/cmr.00072-20 sha: doc_id: 319013 cord_uid: oytqcifa convalescent plasma (cp) therapy has been used since the early 1900s to treat emerging infectious diseases; its efficacy was later associated with the evidence that polyclonal neutralizing antibodies can reduce the duration of viremia. recent large outbreaks of viral diseases for which effective antivirals or vaccines are still lacking has renewed the interest in cp as a life-saving treatment. the ongoing covid-19 pandemic has led to the scaling up of cp therapy to unprecedented levels. compared with historical usage, pathogen reduction technologies have now added an extra layer of safety to the use of cp, and new manufacturing approaches are being explored. this review summarizes historical settings of application, with a focus on betacoronaviruses, and surveys current approaches for donor selection and cp collection, pooling technologies, pathogen inactivation systems, and banking of cp. we additionally list the ongoing registered clinical trials for cp throughout the world and discuss the trial results published thus far. dengue virus, and zika virus) (2) , chikungunya virus (3) , influenza viruses a [e.g., a(h1n1) and a(h5n1)] (4), ebola virus (ebov) (5) , and respiratory betacoronaviruses (sars-cov and middle east respiratory syndrome-cov [mers-cov]), which could put us in situations very similar to the situation with the current pandemic and which require the development of specific intervention protocols. while vaccination strategy is undoubtedly a viable goal, development of a vaccine requires a time frame not compatible with an emergency situation. it is also a prophylactic approach that has no use in the therapeutic setting. on the other hand, the use of antivirals is valuable for the therapeutic setting (6, 7) . for the limited number of antiviral agents currently available, unless provided free of charge to developing countries, financial cost is an issue. additionally, manufacturing is hard to scale up in short time frames. in situations in which the new pathogen is able to induce an immune response with the production of neutralizing antibodies, passive transfusion of convalescent blood products (cbps), in particular, convalescent plasma (cp), has proven to be a winning and logistically feasible therapeutic strategy (8) . cbps can be manufactured by collecting whole blood or apheresis plasma from a convalescent donor. this approach has been used since 1900 (9) , and previous experiences have been reported elsewhere (10) . the main accepted mechanism of action for cbp therapy is clearance of viremia, which typically happens 10 to 14 days after infection (11) . so cbp has been typically administered after the appearance of early symptoms to maximize efficacy. convalescent whole blood (cwb), in addition to antibodies, provides control of hemorrhagic events, as in ebola virus disease, if transfusion occurs within 24 h to maintain viable platelets and clotting factors. nevertheless, cp best fits settings where only antibodies are required. in this review, we have described current technologies for cp collection, manufacturing, pathogen inactivation, and banking of cp. then we have summarized historical settings of cbp application, with a specific focus on applications for covid-19 and other future pandemics. several articles included in this review are available as preprints which have not yet passed peer review, as indicated in the reference section. convalescent donor testing for neutralizing antibodies is mandatory in upstream donor selection. donor selection is generally based on neutralizing antibody titer, as assessed with a plaque reduction neutralization test (prnt) (12) , which requires a viable isolate, replication-competent cell lines, and skilled personnel. since prnt takes time to be set up and requires expensive facilities, in resource-poor settings or in time-sensitive scenarios, collection based on a retrospective prnt or, alternatively, on an enzymelinked immunosorbent assay (elisa) targeting the recombinant receptor binding domains (rbds) of the viral antireceptor has often been implemented; under these circumstances, studies have suggested that elisa ratios/indexes have good correlations with prnt titers; e.g., the euroimmun elisa igg score detected 60% of samples with prnt titers of ͼ1:100, with 100% specificity using a signal/cutoff reactivity index of 9.1 (13) . the current understanding of neutralization suggests that the virus-blocking effect is related to the amount of antibodies against different epitopes coating the virion, whose stoichiometry is in turn affected by antibody concentration and affinity. the donor should preferably live in the same area as the intended recipient(s) to allow consideration of mutations of the target viral antigens. sars-cov-2 s protein has already mutated after a few months of viral circulation (14) , with one mutation outside the receptor-binding motif (23403a¡g single nucleotide polymorphism, corresponding to a d614g amino acid change) currently defining a dominant clade (15) characterized by reduced s1 shedding and increased infectivity (16) . nevertheless, it should be considered that preferring indigenous donors could represent a drawback in areas with epidemics of other infectious diseases (e.g., malaria). three approaches are theoretically available to recruit cp donors, with each having pros and cons. the least cost-effective approach is screening the general regular blood donor population for the presence of anti-sars-cov-2 antibodies. in areas of endemicity, such a strategy provides many fit donors with the additional benefit of seroprevalence study in the general population (80% of cases being asymptomatic) but requires a large budget. alternatively, recruitment of hospital-discharged patients is highly cost-effective (patients can be easily tested before discharge and tracked), but patients who have required hospitalization are highly likely to be elderly with comorbidities and, hence, unfit to donate. the intermediate approach, whenever allowed by privacy regulations, is making calls to positive cases under home-based quarantine to solicit donations; given the large numbers of such cases, some of them are likely to be regular donors, and home-based convalescence suggests that they are fit enough to donate. nevertheless, lessons from mers (17) and preliminary evidence with covid-19 (18) (19) (20) suggest that patients with mild symptoms may develop low-titer antibodies, making antibody titration even more important in the population-wide and home-based approaches. plasma samples collected an average of 30 days after the onset of symptoms had undetectable half-maximal neutralizing titers in 18% of donors (21) . under emergency settings, it has often happened that donors are not screened for high-titer neutralizing antibodies or that low-titer donations are collected; nevertheless, as soon as the urgent requests are satisfied and a buffer stock has been created, repeat donations should preferably focus on donors with high titers (22) . as recently suggested, plasmapheresis could additionally benefit the convalescent covid-19 donor by reducing the prothrombotic state via the citrate-based anticoagulants administered during donation and by removal of high-molecular-weight viscous components (23) . in addition to interventional trials, in the united states several trials have been initiated to create registries (e.g., clinicaltrials.gov registration no. nct04359602) or collect plasma with titers of ͼ1:64 from immune donors for banking purposes, without immediate reinfusion (e.g., trial nct04360278, nct04344977, or nct04344015). these approaches should be encouraged to better face the next waves of the covid-19 pandemic. cp should be collected by apheresis in order to ensure larger volumes than available with whole-blood donations and more frequent donations and to avoid causing unnecessary anemia in the convalescent donor. double filtration plasmapheresis (dfpp) using fractionation filter 2a20 is under investigation as an approach to increase igg yield by 3 to 4 times ( table 1 , trial nct04346589 in italy); since dfpp-derived plasma is not an ordinary blood component but, rather, a discard product, additional regulations could apply in different countries. a very exploratory approach is under investigation in a chinese trial collecting immunoglobulins from convalescent donors by immunoadsorption (trial nct04264858), which could be an alternative to plasma fractionation. although neither the u.s. food and drug administration (fda) (24) nor the european center for disease control (ecdc) is recommending pathogen reduction technologies (prt) for cp (25) , several national authorities consider that, under emergency settings, donor screening and conventional viral nucleic acid testing (nat) (i.e., hiv, hepatitis c virus [hcv] , and hepatitis b virus [hbv] nat) would not be enough to ensure cp safety (12) . under this scenario, additional virological testing and prt approximately double the final cost of the therapeutic dose. several technologies for prt have been approved and are currently marketed. solvent/detergent (s/d)-filtered plasma provides quick inactivation of ͼ4 logs of most enveloped viruses; although the technology was developed and is widely used for large plasma pools, small-scale reduction has been reported. the technology relies on several steps: addition of 1% tri(n-butyl) phosphate-1% triton x-45, elimination of solvent and detergent via oil extraction and filtration, and finally sterile filtration (26) . filtration across 75-to 35-nm-pore-size hollow fibers could remove large viruses (such as betacoronaviruses) while preserving igg (27), but this has not been implemented yet. in recent years photoinactivation in the presence of a photosensitizer has become the standard for single-unit inactivation; approved technologies include combinations of methylene blue and visible light (28) (theraflex), amotosalen (s-59) and uv a (29) (intercept), and riboflavin and uv b (30) (mirasol). these methods do not affect immunoglobulin activity. fatty acids are also an option. in 2002 it was reported that caprylic acid (31) and octanoic acid (32) were as effective as s/d at inactivating enveloped viruses. heat treatment of plasma has been used in the past (33, 34) but comes with a risk of aggregation of immunoglobulins (35, 36) . figure 1 represents how cp and intravenous immunoglobulin (ivig) can be obtained under modern fractionation procedures. as per cp collection, two approaches can be pursued. large-pool products. pharmaceutical-grade facilities typically pool 100 to 2,500 donors to manufacture s/d-inactivated plasma. ivigs are similarly prepared from pools of 2,000 to 4,000 liters of plasma (or 100 to 1,000 liters in the case of hyperimmune ivig) (37, 38) . such volumes can hardly be obtained from cp donors, and timely creation of dedicated cp production chains pose difficult good manufacturing practice (gmp) issues within plasma vendor plants (38) . mpfs into immunoglobulins. in order to be economically sustainable, contract (private-run) fractionation typically requires well over 10,000 liters of plasma per year, and domestic (state-owned) fractionation typically requires over 100,000 to 200,000 liters per year in addition to starting up a fractionation facility. an "on-the-bench" minipool fractionation scale (mpfs) process (5 to 10 liters of plasma, i.e., approximately 20 recovered plasma units) using disposable devices and based on caprylic acid precipitation has been under development in egypt since 2003 and has proved effective at purifying coagulation factors (39) and immunoglobulins (6-fold enrichment) (40) . the same disposable bag system has also been combined with s/d reduction (26) . cp can be either frozen or transfused as a fresh product. aliquots of 200 to 300 ml can be easily achieved from a single unit using modern prt kits. banking cp at temperatures below ϫ25˚c (according to european directorate for the quality of medicines [edqm] or fda guidelines for ordinary plasma for clinical use [41] ) is encouraged in order to produce cp as an off-the-shelf, ready-to-use product. most regulatory systems require that cp be tracked informatically as a blood component different from ordinary plasma for clinical use. the final validation label should report that the donor has tested negative by pcr for the convalescent disorder and additional microbiological tests and should describe the inactivation method. a single cycle of freezing and thawing does not significantly affect the quantity or function of immunoglobulins (42) . given that covid-19 ab blood group recipients can receive cp units only from scarce matched blood group ab donors, to increase the pool of compatible units several authors have recommended titration of anti-a and anti-b isoagglutinins and transfusion of low-titer (ͻ1:32) non-abo-compatible cp units (i.e., o, a, and b) to ab recipients (22, 43) . sars-specific neutralizing antibodies usually persist for 2 years (44), and a decline in prevalence and titers occurs in the third year (45) . convalescent anti-sars immunoglobulins were manufactured on a small scale (8, 46) . three infected health care workers with sars progression despite the best supportive care (bsc) survived after transfusion with 500 ml of cp; viral load dropped to zero at 1 day after transfusion (47). soo et al. reported in a retrospective nonrandomized trial that treatment with cp (titer of ͼ1:160) in 19 patients was associated with a shorter hospital stay and lower mortality than continuing treatment with high-dose methylprednisolone (48) . amotosalen photochemical inactivation of apheresis platelet concentrates demonstrated a ͼ6.2 log 10 mean reduction of sars-cov (49) . theraflex reduces infectivity of sars-cov in plasma (50) . heating at 60°c for 15 to 30 min reduces sars-cov from plasma without cells (51) , while maintaining 60°c for 10 h is required for plasma products (52) . in addition, sars-cov was found to be sensitive to s/d (51, 53) . antibody responses to mers persist for less than 1 year, and the magnitude correlates with the duration of viral rna shedding in sputum (but not with viral load). patients with mild disease have very low antibody titers, making cp collection challenging in mers convalescents (54) . a study reported that only 2.7% (12 out of 443) exposed cases tested positive by elisa, and only 75% of them had reactive microneutralization assay titers (17) . cp with a prnt titer of ն1:80 provides clinical benefit in mers (55) . a case of transfusion-related acute lung injury (trali) following cp transfusion in a patient with mers was reported (56, 57) . mers-cov load in plasma was reduced by theraflex (58), intercept (59), mirasol (60) , and heating at 56°c for 25 min (61); in all cases, passaging of inactivated plasma in replication-competent cells showed no viral replication. as soon as the covid-19 pandemic appeared (62, 63) , several authors suggested cp as a potential therapeutic agent (64, 65) . of interest, the most critically ill patients show prolonged viremia (strongly correlated with serum interleukin-6 [il-6] levels) (10), which makes feasible therapeutic intervention with antiviral agents and immunoglobulins even at late stages. viral shedding in survivors can last as long as 37 days (62), mandating sars-cov-2 rna screening in cp donors. serum igm and iga antibodies appear in covid-19 patients as early as 5 days after symptom onset (66), while igg can be detected at day 14 (67) . iggs are generally detected after 20 days (68, 69) . severely ill female patients generate igg earlier and at higher titers (70, 71) ; the greatest part of the neutralizing antibody response has been shown to be associated with the igg 1 and igg 3 subclasses (72, 73) . duration of anti-sars-cov-2 antibodies in plasma is currently unknown; while the overall antibody responses for other betacoronaviruses typically declines after 6 to 12 months (74), sars-specific neutralizing antibodies usually persist for 2 years (44) . so, in the vast majority of countries, a suitable donor could donate 600 ml of plasma (equivalent to 3 therapeutic doses under most current trials) every 14 days for a minimum of 6 months. up to 7 plasma donations have been proven not to decrease antibody titers in convalescent donors (18) . in contrast to sars and mers patients, most covid-19 patients exhibit few or no symptoms and do not require hospitalization; this could suggest that the majority of convalescent donors are best sought in the general population although specific studies on antibody titers in mildly symptomatic patients suggest low titers (18) (19) (20) . sars-cov-2 is reduced by ͼ3.4 logs by mirasol (75) (and likely by other prts); nevertheless, sars-cov-2 viral rna (vrna) is detectable at low viral loads in a minority of serum samples collected in acute infection but is not associated with infectious sars-cov-2 (76) . intercept treatment has been proven not to reduce sars-cov-2 neutralizing antibody titers (77) . the main contraindications to cp therapy are allergy to plasma protein or sodium citrate, selective iga deficiency (ͻ70 mg/dl in patients 4 years old or older), leading to anaphylaxis from iga-containing cp (78) , or treatment with immunoglobulins in the last 30 days (because of a risk of developing serum sickness). as in many other trial settings, concurrent viral or bacterial infections, thrombosis, poor compliance, short life expectancy (e.g., multiple-organ failure), and pregnancy or breastfeeding are also contraindications (79) . in an early case series from china, five patients under mechanical ventilation (4 of 5 with no preexisting medical conditions) received transfusions of cp with an elisa igg titer of ͼ1:1,000 and a prnt titer of ͼ40 at days 10 to 22 after admission. four patients recovered from acute respiratory disease syndrome (ards), and three were weaned from mechanical ventilation within 2 weeks of treatment, with the remaining patients being stable (80) . another chinese pilot study (chictr2000030046) of 10 critically ill patients showed that one dose of 200 ml of cp with a neutralizing antibody titer of ͼ1:640 resulted in an undetectable viral load in 7 patients, with radiological and clinical improvement (81) . a third series of 6 cases with covid-19 pneumonia in wuhan showed that a single 200-ml dose of cp (with titers of anti-s antibodies determined by chemiluminescent immunoassay [clia] only) administered at a late stage led to viral clearance in 2 patients and radiological resolution in 5 patients (82) . pei et al. reported successful treatment of 2 out of 3 patients with 200-to 500-ml doses of cp (83) . recovery from mechanical ventilation was also reported by zhang et al. in a single patient after antibodies in cp were titrated with an anti-n protein elisa (84) . no improvement in mortality despite viral clearance was reported in a retrospective observational study recruiting 6 late-stage, critically ill patients treated with gold-immunochromatographytitrated cp, compared to results in 13 untreated controls (85) . one case of recovery in a centenarian patient who received 2 cp units (s-rbd-specific igg titer of ͼ1:640) was also reported (86) . many more case reports and small case series are accumulating in the literature; successful treatment was reported in 3 cases with ards and mechanical ventilation using two 250-ml cp doses (titrated with elisa only) in south korea (43, 87) , in 2 cases from iraq (88), in 8 out 10 severe cases from mexico (89) , in 20 out of 26 severe cases from turkey (90), in a kidney transplant recipient from china (91) , in a case with severe aplastic anemia in poland (92) , in a case with x-linked agammaglobulinemia in spain (93) , and in 1 patient with marginal-zone lymphoma treated with bendamustine and rituximab in the united kingdom (94) . centers in the united states reported successful treatment with cp in 18 out of 20 patients in a series (95) , in 27 out of 31 patients with severe to life-threatening disease in another series (96) , in one case with myelodysplastic syndrome (97) , in a critically ill obstetric patient (in combination with remdesivir) (98) , and in an allogeneic stem cell transplant recipient (99) . in a single-arm phase ii trial (nct04321421 [100] ) run in lombardy, 49 patients with moderate to severe disease were treated with up to 3 units of prt-treated cp (250 to 300 ml/48 h) having neutralizing antibody titers of ն1:160 in 96% of cases. importantly, the viral inoculum was 50 50% tissue culture infective doses (tcid 50 ) instead of the usual 100 tcid 50 . seven-day mortality was 6% versus 16% in a historical cohort. one case of trali was reported (101) . in a large case series from wuhan, 138 patients were transfused with 200 to 1,200 ml of cp at a median of 45 days after symptom onset and experienced a 50% lower intensive care unit (icu) admission rate and mortality than the group treated with best supportive care. responders had higher lymphocyte counts, lower neutrophil counts, and lower lactate dehydrogenase (ldh), type b natriuretic peptide (bnp), urea nitrogen, procalcitonin, glucose, and c-reactive protein (crp) levels. complete data on neutralizing antibody titers in covid-19 convalescent plasma (ccp) units were not available, but responders tended to have received cp units with higher antibody levels (102) . in the first retrospective, randomized controlled trial published to date, 39 patients in new york with severe covid-19 were transfused with 2 units of abo-type matched cp with anti-spike antibody titers of ն1:320 (measured by a two-step spike proteindirected elisa). cp recipients were more likely than control patients to not increase their supplemental oxygen requirements by posttransfusion day 14 (odds ratio [or], 0.86), but survival improved only for nonintubated patients (hazard ratio [hr], 0.19) (103) . another prospective, multicenter randomized controlled trial from china (chictr2000029757) enrolled 103 patients with severe to life-threatening covid-19. the study was underpowered because of earlier than expected (200 cases) termination. cp (9 to 13 ml/kg from donors with s-rbd igg titer of ն1:640) was associated with a negative sars-cov-2 pcr test at 72 h in 87.2% of the cp group versus 37.5% of the bsc group, but clinical improvement at 28 days was statistically different only in patients with severe, but not in life-threatening, disease (104) . table 1 lists the other ongoing cp trials in covid-19 patients collected from different web portals. the united states has developed a specific platform for facilitating clinical trials (https://ccpp19.org/), while the international society of blood transfusion created a resource library (https://isbtweb.org/coronaoutbreak/covid-19 -convalescent-plasma-document-library/). at the same time, in the united states an expanded-access program (eap) has been approved by the fda and coordinated by mayo clinic and has led to treatment of more than 30,000 patients as of 8 july 2020 (https://www.uscovidplasma.org). a preliminary report on the first 20,000 patients (66% from intensive care units) confirms safety (ͻ1% severe adverse events and 14.9% mortality at 14 days) and suggests a benefit compared to results with historical cohorts, especially if cp is administered before mechanical ventilation (105, 106) ; donor titers were not disclosed, and evidently some donations were not titrated before reinfusion. largely similar data have been reported from a 25-patient case series from houston, texas, where cp has been used as an emerging investigational new drug (eind) (107). typically, 1 or 2 doses of 200 ml are administered (if 2 doses are used, they are administered at least 12 h apart), with infusion rates of 100 to 200 ml/h. the cumulative dose should be targeted according to body weight and antibody titer (22) . several authors have suggested plasma exchange with cp (i.e., high-volume therapeutic plasmapheresis followed by cp transfusion) rather than cp transfusion alone in order to clear proinflammatory cytokines from the bloodstream (108, 109) , and several successful case reports deploying nonconvalescent plasma have been reported (110) (111) (112) . one randomized controlled trial (nct04374539) is ongoing in patients with severe covid-19, but unfortunately no trial to date is testing plasmapheresis followed by cp. unfortunately, most trials in westernized countries (in contrast to ones ongoing in china) have no control arm, which will impair efficacy interpretation. when present, the control arm consists of the best supportive care alone (typically oxygen and hydroxychloroquine at 400 mg twice a day [b.i.d.] for 10 days) or combined with intravenous placebo or standard (nonconvalescent) plasma (eventually of pharmaceutical grade). since other plasma components (e.g., aspecific immunoglobulins or isoagglutinins; see below) could contribute to clinical benefit, the latter approach is ideal for dissecting the specific contribution of neutralizing antibodies although concerns could be raised by the prothrombotic nature of covid-19 pathology (see side benefits from cp in covid-19, below). even using a placebo control in late-stage patients (refractory to former lines) could pose some ethical concerns because it denies treatment opportunities to an unresponsive disease. future trials should investigate combined antiviral and cp therapies. notably, several plasma manufacturers are attempting to develop sars-cov-2specific hyperimmune sera (e.g., takeda's tak-888 merged with biotest, bpl, lfb, octapharma, and csl behring into the convalescent plasma coalition [113] ; kedrion and kamada have joint ventures [81] ). cp is considered an experimental therapy, and, as such, phase 3 randomized controlled trials should be encouraged. despite this recommendation, in emergency settings phase 2 trials are usually started, hampering efficacy analysis. response in published trials is generally measured clinically (pao 2 /fio 2 ratio) or radiologically according to target organs. nevertheless, surrogate endpoints can include anti-sars-cov-2 antibody titer or absolute lymphocyte count increases in recipients, as well as decreases in recipients' sars-cov-2 viral load or il-6 levels. whenever quantitative pcr is not available, cycle threshold (c t ) value increases in qualitative pcr after transfusion could be a proxy for reduced viral load. the first concern is transfusion-transmitted infection (tti (114) or by some individuals. in addition, there is a now a recognized risk of hepatitis e the within the u.k. blood donor population (115) , most likely due to the consumption of poorly cooked pork products (116, 117) , for which screening has only relatively recently been initiated (71) . although this does not preclude such sars-cov-2 convalescent plasma/serum from being used therapeutically within the united kingdom, these other risks should be considered during larger clinical trials or with compassionate use in individual patients. respiratory betacoronaviruses produce only a mild and transient viremia. with sars-cov, limited replication in lymphocytes (118) leads to significant risk only for recipients of blood products with high concentrations of donor lymphocytes (peripheral blood stem cells, bone marrow, granulocyte concentrates, etc.). preliminary reports have shown that sars-cov-2 viremia persists only in critically ill patients (10) . the second concern is trali, which can be life-threatening in patients who are already suffering from ali. male donors are usually preferred in order to avoid the risk of transfusing anti-hla/hna/hpa antibodies from parous women. in the case of covid-19, where female patients have been shown to have higher igg levels, this could be detrimental, and anti-hla/hna/hpa antibody screening could be implemented. antibody-dependent enhancement (ade) is also a theoretical concern related to passive or active antibodies (targeting s protein domains other than the rbd) facilitating igg-coated virion entry into macrophages via fc␥ receptors and/or complement receptors (119, 120) , leading to activation of the rna sensing toll-like receptors (tlr) 3, 7, and 8 and finally to elevated production of tumor necrosis factor (tnf) and il-6 (a so-called cytokine storm). elisas discriminating the difference between total and rbd-binding antibodies could be useful to inspect the occurrence of ade. genetic polymorphisms (e.g., fc␥riia [121] ) can also contribute to ade. to date, potential evidence supporting a role for ade in covid-19 include the following: (i) the correlation between disease severity and total anti-sars-cov-2 antibody levels (70, (122) (123) (124) , including neutralizing antibodies (125, 126) ; (ii) the low prevalence of symptoms in covid-19 patients younger than 20 (who have likely not been primed by infection with the other common cross-reacting coronavirus 229e or oc43 or anyway have low-affinity anti-coronavirus igg [127, 128] ); (iii) the occurrence, in sars, of ade at low antibody titers in vitro (129) and correlation in patients of high igg titers and early seroconversion with disease severity (130) . overall, these findings raise concerns for usage of low-titer cp units (131) . other evidence is the high level of afucosylated igg against s protein, facilitating fcr binding, that is produced in the most severely ill patients (132, 133) . a last, covid-19-specific, concern is worsening of the underlying coagulopathy (134) from clotting factors in transfused plasma (not only cp but also nonconvalescent plasma in control arms); since this has not been reported to date, it remains a theoretical concern. obviously, patients with humoral immune deficiencies can benefit from polyclonal antibodies contained in cp, and patients with hemorrhagic diathesis can benefit from clotting factors. plasma is also likely to contain antibodies against other common betacoronaviruses associated with the common cold, which have been shown to cross-react with sars-cov-2 antigens in intravenous immunoglobulin (ivig) preparations (135) , likely stemming from recent infection with another human betacoronavirus (128) . accordingly, ivig led to clinical and radiological recovery in 3 chinese patients with severe covid-19 (136) , and the same team is now leading a randomized controlled trial (nct04261426). after demonstration that blood group o health care workers were less likely to become infected with sars-cov (137), a research group proved that anti-a blood group natural isoagglutinins (which can also be found in cp plasma from blood group o and b donors) inhibit sars-cov entry into competent cells (138) . such binding could opsonize virions and induce complement-mediated neutralization (139) . since sars-cov-2 uses the same receptor as sars-cov, anti-a isoagglutinins are expected to have similar effects against sars-cov-2 (140); accordingly, clusters of glycosylation sites exist proximal to the receptor-binding motif of the s protein from both sars-cov (141) and sars-cov-2 (142) . several publications showed that the odds ratio for acquiring covid-19 is higher in blood group a than in blood group o (143) (144) (145) (146) (147) , and one showed the abo gene polymorphism to be the most significant at predicting severity of covid-19 (147) . covid-19 has more severe clinical presentations and outcomes in the elderly and in males; intriguingly, elderly males are known to experience reductions in isoagglutinin titers (148, 149) . although alternative explanations exist (150, 151) , studies are hence ongoing to evaluate correlations between isoagglutinin titers and outcomes in blood group o and b patients (152) . if the correlations are confirmed, while preserving abo match compatibility, blood group o and b donors for cp in covid-19 could be preferred, and their anti-a isoagglutinin titers should be tested. cp manufacturing should be considered among the first responses during a pandemic while antivirals and vaccines are tested. despite huge competition from trials employing small molecules, multicenter randomized controlled trials should be encouraged in order to establish efficacy and provide hints about the most effective schedule (timing and dose). coronavirus disease 2019 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isohemagglutinin titers and sars-cov2 neutralization: implications for children and convalescent plasma selection ebola virus convalescent blood products: where we are now and where we may need to go focosi is a hematologist employed as resident transfusion physician at the largest blood bank in italy since 2009. he has been a transplant immunologist and immunogeneticist, quality assurance manager, and production manager. he has received awards from the european federation of immunogenetics, the european society of organ transplantation, and the italian society of hematology. he has a ph.d. degree in clinical and fundamental virology, and a master's degree in clinical trials. he has authored 124 articles indexed in pubmed, for a global h-index of 24, on topics ranging from emerging viral infections to new markers of immune competence. julian w. tang is a hospital consultant and medical virologist, with special interests in the diagnosis, treatment, epidemiology, and infection control of influenza and respiratory viruses, congenital viral infections, hiv, and blood-borne viruses. he also has a ph.d. in zoology. he has formerly been associate professor at the university of alberta and assistant professor at the chinese university of hong kong.marco tuccori is a clinical pharmacologist with special interest in pharmacovigilance and pharmacoepidemiology. he also has a ph.d. in pharmacology and medical physiology. he is currently pharmacovigilance manager at the unit of adverse drug reactions monitoring of the university hospital of pisa and coordinator of the tuscan regional centre of pharmacovigilance. he collaborates with the agenzia italiana del farmaco (aifa) as a member of the working group for signal detection analysis on drugs and vaccines. he was a member of the advisory board (formerly executive committee) of the international society of pharmacovigilance (isop) from 2012 to 2019. he is the author of about 90 articles in peer-reviewed scientific journals and four chapters of books. key: cord-321697-yua3apfi authors: crigna, adriana torres; samec, marek; koklesova, lenka; liskova, alena; giordano, frank a.; kubatka, peter; golubnitschaja, olga title: cell-free nucleic acid patterns in disease prediction and monitoring—hype or hope? date: 2020-10-29 journal: epma j doi: 10.1007/s13167-020-00226-x sha: doc_id: 321697 cord_uid: yua3apfi interest in the use of cell-free nucleic acids (cfnas) as clinical non-invasive biomarker panels for prediction and prevention of multiple diseases has greatly increased over the last decade. indeed, circulating cfnas are attributable to many physiological and pathological processes such as imbalanced stress conditions, physical activities, extensive apoptosis of different origin, systemic hypoxic-ischemic events and tumour progression, amongst others. this article highlights the involvement of circulating cfnas in local and systemic processes dealing with the question, whether specific patterns of cfnas in blood, their detection, quantity and quality (such as their methylation status) might be instrumental to predict a disease development/progression and could be further utilised for accompanying diagnostics, targeted prevention, creation of individualised therapy algorithms, therapy monitoring and prognosis. presented considerations conform with principles of 3p medicine and serve for improving individual outcomes and cost efficacy of medical services provided to the population. liquid biopsy (lb) and individualised profiling of biomarker patterns presented in body fluids represent a revolutionary approach in the workframe of 3p medicine [1] . current paper is dedicated to the liquid biopsy utilising specifically blood samples as the best explored source of information amongst other sorts of body fluids [2] . in the last years, cell-free nucleic acids (cfnas) "signature" attracted a lot of attention for diagnostic and treatment purposes. altered profiles of cfnas have been detected under physiological conditions, e.g. by making sport, suboptimal conditions such as overtraining syndrome in physical exercises [3] , acute and chronic pathological conditions including sepsis, stroke, trauma, myocardial infarction, autoimmune diseases and cancers [4] . to this end, certainly the area of oncological research is particularly advanced implementing ctdna and mirna detection and quantification for diagnostic and treatment purposes [5] . adriana torres crigna and marek samec contributed equally to this work. however, independently of the application area, the main goal remains the same, namely to look for pathology-specific patterns [6] [7] [8] as well as for patterns clearly indicating associated risks, for example, in vasospastic individuals who may be particularly predisposed to an increased stress sensitivity [9] [10] [11] , neuro/degenerative pathologies [12, 13] and/or aggressive metastasing cancers [14, 15] . diagnostic and prognostic potential of cell-free nucleic acids' signature in stress conditions and stress-related pathologies dysregulation at the level of cfnas acts as a promising diagnostic biomarker panel for measuring imbalanced stress and for predicting stress-associated pathologies. according to the world health organisation (who), stress presents the epidemic of the third millennium [16] . accumulated evidence suggests a tight association between chronic stress and psychiatric disorders [17] [18] [19] [20] [21] . especially severe, prolonged and/ or chronic stress of any origin such as exercise-induced oxidative stress [22] (see "physical activity and exercise-induced oxidative stress" section), hormonal stress [23] , emotional stress and psychological burden [24] [25] [26] [27] as well as metabolic stress, e.g. in diabetes mellitus [28, 29] (see also below "association between diabetes mellitus and carcinogenesis: diagnostic and therapeutic potential of cell-free nucleic acids" section) and hyperhomocysteinaemia [30, 31] amongst others, is associated with highly increased ros production and insufficient repair capacity-both linked to oxidative damage of mitochondria and consequent mitochondrial dysfunction leading to the development of cardiovascular impairments [32] [33] [34] , neuro/degenerative pathologies [34] [35] [36] [37] , impaired healing [34] and malignant cell transformation [34, [38] [39] [40] [41] [42] . noteworthy, the pathomechanisms carry a systemic character [43] that is crucial for tracing corresponding alterations in a minimally invasive manner utilising blood samples and other body fluids [1] . an application of liquid biopsy is a promising approach to identify biomarker patterns specific for stress and stressassociated diseases. prominent examples are summarised below. acquired data revealed lower expression of serum mir-183 and mir-212 in major depressive disorder (mdd) patients after antidepressant therapy [44] . further, mir-16, mir-135a and mir-1202 were significantly reduced in serum of patients diagnosed with depression compared with healthy individuals [45] . plasma mir-134 (associated with the regulation of synaptic plasticity and neurogenesis) was downregulated in a cohort of patients with mdd compared with healthy controls. measurements of mir-134 patterns are also useful to distinguish between mdd, bipolar disorder and schizophrenia [46] . further, an increased expression of mir-124-3p has been detected in serum of antidepressant-free mdd patients compared with healthy controls [47] . another study revealed significantly higher levels of plasma mir-451a and lower levels of mir-320 in a group of depressed patients [48] . another study detected significantly higher levels of serum mir-221-3p, mir-34a-5p and let-7d-3p in patients with mdd compared with controls [49] . moreover, depressive symptoms were associated with the downregulation of plasma mir-144-5p considered as a useful biomarker for pathological processes associated with depression [50] . posttraumatic stress disorders (ptsds) as a consequence of acute traumatic stress demonstrate specific patterns of mir-142-5p, mir-19b, mir-1928, mir-223, mir-332, mir-324, mir-421-3p, mir-463 and mir-674. anxiety and delayed fear are reflected in specific patterns of the panel comprising mir-142-5p, mir-1928, mir-223 and mir-19b [51] as detected, for example, in veterans suffering from ptsds. to this end, mir-203a-3p derived from extracellular vesicles was upregulated, whilst mir-339-5p was downregulated in a cohort of ptsds patients compared with controls [52] . differentially expressed circulating mirnas associated with ptsds were detected in another study focused on stress-related disorders in the population of military veterans [53] . in a preclinical study, mir-24-2-5p, mir-27a-3p, mir-30e-5, mir-3590-3p, mir-532-5p and mir-362-3p patterns were decreased in rats with manifested vulnerability to chronic stress, whereas another panel comprising mir-28-3p, mir-139-5p, mir-326-3p and mir-99b-5p was downregulated in rats more resistant to stress-both compared with controls [54] . in the context of stress, cfdna is an excellent biomarker candidate for clinical application considering circulating cellfree mitochondrial dna (ccf-mtdna). correlation between serum ccf-mtdna and psychological stress was demonstrated in the study focused on the cohort of healthy midlife adults. a brief psychological challenge in tested volunteers led to increased serum ccf-mtdna, in contrast to circulating cellfree nuclear dna [55] . increased plasma concentrations of ccf-mtdna have been demonstrated also for patients diagnosed with mdd and concluded as a biomarker associated with psychiatric disorders and useful for monitoring the pathology development and therapy response [56] . table 1 summarises cfnas associated with stress. adapted exercise has an ability to inhibit ros production, ameliorates the antioxidant capacity and improves mitochondria efficiency reducing oxidative stress and cellular damage [59] . temporary increased levels of inflammation and cfdna were observed in various acute exercises such as marathon, ultramarathon, resistance exercise, continuous, interval, and incremental treadmill running, and incremental rowing exercise [60] [61] [62] [63] . however, during the period of physiologic recovery, the cfdna levels usually come back to the baseline level [64] . in contrast, overtraining causes exercise-induced oxidative stress [22] . consequently, the question is-how to distinguish between beneficial physical activity on one hand and damaging exercise-induced oxidative stress on the other hand, when providing recommendations at individual level? circulating cfnas might be helpful answering this question, since their patterns strongly depend on the intensity and duration of exercise being complementary to specific metabolic markers such as lactate and creatine kinase recognising muscle damage [3, 65] . to this end, the overtraining and induced inflammation are well reflected in c-reactive protein (crp) levels as the marker of inflammation and highly increased concentration of plasma cfdna in proportion to training load [66] . in addition, there is no any significant difference in circulating cfdna between obese and normal-weight subjects [67] . noteworthy, although remaining unchanged in its absolute quantity, the proportional input by the foetal cfdna is reduced in mother's blood by increased concentration of cfdna linked to the exercise during and immediately after the physical activity. this proportion is normalised by 30 min after the exercise is finished [68] . the initiative called education outside the classroom (eotc) promoting physical activity against obesity in youth, has demonstrated increased level of cfdna for bothsedentary behaviour and moderate-to-vigorous physical activity groups. based on the results, the authors recommend light physical activity with the best potential to be supportive for health in examined children [69] . further, diabetes predisposition can be diagnosed, e.g. in persons with sedentary lifestyle by applying mir-192 and mir-193b panel detected in the prediabetic stage but not in diabetic patients. moreover, in glucose-intolerant mice and prediabetic individuals, regular exercises as a therapeutic strategy have normalised the mirna patterns to the baseline level [70] . furthermore, in healthy subjects, 74 circulating mirnas associated with various heart diseases were evaluated at baseline, immediately after exercise and after 24 h. only mir-103a-3p was reduced in both types of exercises: 10 km and marathon races. furthermore, increased serum levels of mir-132-3p and mir-150-5p were detected forthwith after the 10km race. on the contrary, decreased serum levels of mir-103a-3p, mir-590-5p and mir-139-5p were observed in the same type of exercise. moreover, decreased levels of mir-103a-3p and mir-375-5p were observed immediately after marathon race ccf-mtdna serum participants (n = 50) exposed to brief psychological challenge ↑ ccf-mtdna [55] ccf-mtdna plasma individuals (n = 50) with mdd versus disease-free controls (n = 55) ↑ ccf-mtdna [56] and remained low also after 24 h. further, several cardiac markers were upregulated and lasted for 24, 48 and/or 72 h after both exercises. taken together, circulating mirnas can be useful for patients with dysfunction symptoms after an acute attack of endurance physical activity [71] . additionally, increased levels of circulating mirnas including mir-126, mir-130b, mir-221 and mir-222 at baseline levels were detected in obese versus normal-weight subjects. these patterns but at higher levels were observed after acute aerobic exercise in obese subjects, even after controlling for vo 2max and insulin resistance (homa-ir) [72] . in summary, specific cfnas patterns have been demonstrated in relationship to physical activity that allows to clearly differentiate between beneficial physical activity and exercise-induced oxidative stress and to provide accompanied diagnostics and individualised recommendations for healthy individuals and athletes, individuals in suboptimal health as well as for a variety of patients. corresponding information is summarised in table 2 . stroke is one of the leading and preventable causes of sudden death and the most common cause of long-term disability worldwide [73, 74] . ischemic stroke (is) accounts for approximately 80-85% of stroke cases against haemorrhagic one [75, 76] . in short, is is associated with a cascade of events including cerebral ischemia, obstructions in cerebral blood flow, generation of reactive oxygen species, inflammatory processes, neuronal damage and apoptosis leading to neurological dysfunction [77] . is is a heterogeneous, multifactorial disease associated with an interaction between genetic and modifiable risk factors [74] . besides evident genetic predisposition, dietary patterns and lifestylerelated stressors strongly contribute to the development of is [78] . current diagnostic approaches applied for is are not rarely associated with some obstacles such as prolonged time of the imaging performance, poor sensitivity and / or data interpretation, particularly in case of asymptomatic clinical picture [78] . to this end, so-called young stroke-the rapidly increasing patient cohort below 50 years of age with unclear aetiology-is particularly challenging for healthcare globally [13] demanding innovative solutions in the framework of 3p medicine. phenotyping and blood-based biomarkers are currently under extensive consideration for the risk assessment and predictive diagnosis of is [13, 79] . to this end, the blood-brain barrier may prevent releasing brain-specific molecules into the bloodstream [80] . however, due to ischemia-related progressive cell death and consequent blood-brain barrier breakdown, the cfdna release into the blood might accompany is [77] . moreover, due to chronic systemic effects, e.g. in vasospastic individuals predisposed to is [13] , a significant increase in the cfdna blood concentration may happen days and weeks before the acute is event. indeed, the cfdna concentration correlates well with the severity at admission and with individual outcomes in is patients [81] supporting meaningful measurements of plasma nuclear and mitochondrial cfdna patterns [82] including specificity of the dna fragmentation (300-400 bp range) profiling for diagnostic and prognostic purposes [77, [81] [82] [83] . mirna panels provide complementary information in overall is diagnostics: circulating exosomal mir-223 is significantly increased in acute is against healthy controls, and its level correlates with stroke severity and individual outcomes [84] . in contrast, serum mir-221-3p and mir-382-5p patterns are downregulated in is patients against healthy controls [85] . moreover, the combination of mir-21-5p and mir-30a-5p was demonstrated as being of great utility to distinguish between hyper-acute, subacute and recovery phase of is [80] . the mirna panel comprising pc-3p-57,664, pc-5p-12,969, mir-122-5p and mir-211-5p demonstrates a correlation between upregulation in is patients and post-mortem is-brain specimens [86] . table 3 summarises information on cfnas in is. liquid biopsy application to early cancer detection demands highly sensitive detection methodology in order to track circulating tumour dna (ctdna) amounts or diverse sub/ cellular structures secreted by precancerous lesions and/or at initial stages of cancer. for instance, testing viral sequences related to tumours, such as human papillomavirus (hpv) or herpesvirus family (epstein-barr virus (ebv), cytomegalovirus (cmv) [87] ), along with ctdna methylation analysis is instrumental for diagnosing hpv-derived precancerous lesions. hpv 16 and 18 strains have been described as related to high risk, potentially leading to cervical cancer [88] . a cervical precancerous state is characterised by changes in collar cells making them more susceptible to cervix cancer development within 10-year time span, if not treated in a timely manner [89] . a meta-analysis study showed that despite the existing heterogeneity amongst studies, hpv cdna detection is a specific and relatively sensitive tool for cervical cancer diagnosis [89] . another study revealed the presence of hpv in 98.4% of tumours, being hpv16 and 18 dependent for 89.4% of cervical cancer patients detected [87] . likewise, rna-seq database indicated the presence of hpv in the majority of cervical cancers [90] . in addition, ebv, cmv and herpesvirus 6 (hhv6) were detected in 21 and 18% of rectal and colon cancers, respectively. ebv was found to be associated with 23% of stomach cancers. herpesviruses are often detected in stomach, colon and rectum cancers. some types of liver cancers have been linked with hepatitis b and c virus (hbv and hcv). hpv was present in a small amount of bladder cancers along with a subset of head and neck cancers [87] . premalignant neoplastic lesions, in particular, adenomas have often been detected to have distinct mirna expression patterns. in a study assessing mirna expression profiles of crc and adenomas mir18a was upregulated in adenoma patients versus healthy controls [91] . a further study revealed ratios amongst three circulating mirna to allow discriminating between benign prostate adenoma (hyperplasia) and prostate cancer in a more specific manner than standardised prostate-specific antigen (psa) levels [92] . another study described the use of several non-invasive biomarkers concomitantly (psa together with androgen receptor cag analysis and promoter methylation analysis) increasing predictive power of the prostate cancer and allowing its discrimination from benign prostate hyperplasia in 70-80% of cases [93] . furthermore, quantitative and qualitative cfdna characterisation has been described as capable to detect certain cancer types [94, 95] , although being challenging yet for cancer screening application [96] , since cfdna origin, specificity and release kinetics have still to be clarified [97] [98] [99] [100] . plasma levels of short and long fragmented dna and total cfdna in oral cancer and precancerous lesions were evaluated and 10-km race (half-marathon) and marathon 10-km race: circulating mir-126, mir-130b, mir-221, mir-222 plasma n = 24 (12 normal-weight and 12 obese) subjects 30-min aerobic exercise (75% vo 2max ). after acute aerobic exercise in obese subjects: ↑ mir-126, ↑ mir-130b, ↑ mir-221, ↑ mir-222 [72] eotc = education outside the classroom, crp = c-reactive protein, t1, t2, t3 = time poins, il-8 = interleukin 8 quantified. results demonstrated an increased cfdna concentration and integrity of dna in oral cancer compared with other cohorts, rendering it a tool for early oral cancer detection [101] . further study evaluated somatic circulating mutations in patients with breast, lung, colorectal and ovarian cancers to assess cancer disease staging [102] . data revealed overall significant increase of cfdna in cancer patients' plasma compared with healthy subjects. thereby, breast cancer cohort demonstrated the lowest mutant allele fraction of ctdna. noteworthy, advanced disease stages iii and iv correlated with higher amount of ctdna compared with early disease stages i and ii across all cancer cohorts [102] . chronic inflammation (together with infectious diseases related inflammation) is estimated to be responsible for approximately 25% of all cancer cases [103] . in the context of inflammatory milieu, epithelial and inflammatory cells secrete reactive oxygen and nitrogen species (ros and rns) causing dna damage [104] . this dna damage and mutagenic lesions, such as 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodg) and 8-nitroguanine, occur in organs undergoing inflammation, eventually driving carcinogenesis [105] . furthermore, parasites, viruses (hpv, ebv and hepatitis virus) and bacteria are considered to be pathogenic agents carcinogenic to humans [105] . inflammation may also be promoted by physical, chemical and immunological factors [103, 106] . chronic inflammation induces tissue injury, due to genetic and epigenetic aberrations, nucleic acid, lipid and protein damage via to ros/rns production. this tissue damage may activate tissue regeneration resulting from stimulation of progenitor/stem cells. thus, accumulation of mutations in stem cells by ros/rns may result in mutant stem cells or cancer stem cells leading to carcinogenesis [105] . consequently, detection of specific cfdna, mirna and methylation patterns are considered of great clinical utility for early cancer detection [107] . indeed, cfdna is known to accumulate under chronic inflammation, due to decreased clearance [108] . cfdna, nuclear and mitochondrial dna are actively secreted and mediate many processes such as immunomodulation, tumour growth progression and inflammation [108] . for instance, prostate carcinogenesis and disease progression are known correlation between upregulation in ischemic stroke patients and post-mortem ischemic stroke-brain specimens [86] nihss national institutes of health stroke scale to be promoted by chronic inflammation [109] [110] [111] . risk factors related to prostatic inflammation are frequently related to immunological, genomic and environmental factors such as physical trauma, urinary microbial infection, chemical irritation, unhealthy diet and abnormal body weight [112, 113] . recruitment of leukocytes, namely macrophages, lymphocytes, granulocytes and monocytes to the prostate have been observed in the prostate cancer driven inflammation responses [114, 115] . in advanced stages of prostate cancers, elevated peripheral blood neutrophil-to-lymphocytes ratios were observed, portraying worse overall survival (os) and reduced sensitivity to chemotherapy and to anti-androgens [116] . although analysis of solid tumour tissues is a golden standard in oncology [117] , tissue biopsies entail some risks for patients apart from being limited in identifying genetic heterogeneity or tracking neoplasm evolution alternations within a tumour [118] . clinical and laboratory advances have broadened tumour-related diagnosis, prognosis and predictive measures. in fact, the use of cfdna has marked a potential minimally-invasive alternative option for genomic diagnostics. ctdna was described to be the tumour-derived fraction of cell-free dna secreted into the blood [119] . ctdna patterns in blood are considered as being a potent analytical option alternative to solid tumour biopsies for cancer detection and monitoring, due to rapid, non-invasive and cost-effective biomarker identification [120] . besides cfdna/ctdna, malignancy-related blood patterns include circulating mirna, circulating tumour cells (ctcs) and exosomes [121, 122] . notably, also saliva, cerebrospinal fluid (csf), pleural fluid, urine and tears are prospective sources of tumour-originated material [123] [124] [125] [126] [127] . ctdna detection in a broad range of neoplasms ctdna, released by cancer cells, have been identified in a broad range of neoplasm types both in early and late cancer stages, displaying levels from < 1 to > 100,000 mutated dna fragments per ml of plasma. in cancer patients, ctdna fractions differ greatly, fluctuating from less than 0.1% to more than 90% of overall cfdna. there is an obvious great variability amongst ctdna detected in patients with differing cancer type; however, variations in ctdna fraction amongst patients with analogous tumour type may be attributed to biological disparities, as well as varying cell death rates within tumour cells [128] [129] [130] . even though there are studies describing a correlation amongst the amount of cfdna, cancer status and disease progression [131, 132] , others reveal cfdna quantification to be insufficient as an independent diagnostic tool, lacking information about tumour development [133, 134] . cfdna low circulation concentration together with its considerable proportion of fragmentation make it a challenging compound to analyse [135] . furthermore, identification and evaluation of ctdna within total cfdna represent a great challenge in cancer detection [128] [129] [130] . nevertheless, ctdna bears the tumour specific molecular features capable of early cancer diagnosis and prediction as well as disease prognosis. ctdna patterns advance diagnostic approach ctdna has been detected in cancer patients' plasma prior to mainstream screening methods: mutation of kras2 and p53 in healthy subjects are described as related to an increased risk of developing bladder cancer within a period of 6 years [136] . detection of plasma/serum dna alterations at early tumour stages along with the current available markers renders ctdna a useful diagnostic mean for early breast cancer [133] . similarly, the quantification of dna levels and microsatellite alterations in plasma dna of lung cancer patients, suggested a correspondence with their clinical condition, serving additionally as non-invasive follow up assays [134] . in a metastatic breast cancer study a decrease in cfdna integrity along with an increase in plasma cfdna concentration has been described compared with healthy controls [137] . another study analysed crc patient samples in a quantitative and qualitative manner. results revealed high plasma and serum cfdna values at the time of surgery, which further increase in relapsed patients, confirming crc and determining cancer status [94] . the ability of tracking therapy response is one of the most significant traits of liquid biopsies, particularly in therapies with resistance mechanisms. kras mutations in colorectal tumour (crc) progression are associated with acquired resistance and reduced response to anti-epidermal growth factor receptor (egfr) therapies [138] . in a study comparing kras and braf mutations in both metastatic crc plasma cfdna and tumour tissue, specificity and sensitivity of 100% for braf v600e mutation and 96% on kras point mutations have been demonstrated. thus, this study reveals high potential for developing better personalised medical services [139] . furthermore, kras mutations were analysed in crc samples, aiming to determine prevalence of kras amplification and evaluate its overall sensitivity to egfr therapies. in presence of this genetic lesion, a lack of responsiveness to anti-egfr inhibitors was found [138] . similarly, another independent study revealed kras mutations to be common determinants of acquired resistance in crc cancer patients [140] . multiregional and shotgun sequencing of circulating tumour plasma dna has revealed the potential to assess molecular heterogeneity of overall disease [141] . in a study quantifying ctdna from crc patients, ctdna determination could track tumour dynamics in patients subjected to chemotherapy or surgery, revealing a potential customisable genetic approach [128] . similarly, ctdna was detected in 97% of patients with metastatic breast cancer with somatic genomic modifications, and identified tumour dynamics greater than ca 15-3 or ctc [129] . another study testing prostate cancer plasma samples determined the genomic scenario and disease progression through the analysis of ctdna in a non-invasive manner [142] . circulating tumour dna patterns have been described as a non-invasive biomarker able to detect marginal disease residues after surgery or neoplastic therapies [143] [144] [145] [146] . detection of ctdna at time of diagnosis in nsclc patients together with residual ctdna is associated with poor prognosis [147] . furthermore, a study revealed the prognostic capacity of ctdna in plasma from crc patients to determine survival rates and increased patient recurrence [148] . elevated levels of cfdna and plasma mutant kras levels (pmkras) were described to be directly correlated, making plasma cfdna to an alternative prognosis biomarker [149] . similar data were published by dawson et al. for the breast cancer patient cohort [129] . in an advanced non-small cell lung cancer (nsclc) patient study, ctdna appeared to be more sensitive to mutation detection than ctc [150] . an independent study also indicated ctdna to be a potent prognostic biomarker [151] . measuring plasma or serum ctdna profiles to monitor cancer development is a promptly developing field with great clinical potential. studies focused on ctdna as a tool for cancer diagnostic, prediction and/or prognosis are summarised in table 4 . ctdna analysis may reinforce its use as personalised treatments for cancer patients. nevertheless, validating studies are essential to bring this tool into daily clinical practice. anomalous mirna patterns have been correlated with pathogenicity of several human cancers [187] . overexpression of mirna in cancer prompts their action as tumour suppressors or oncogenes depending on the target [188] ; some mirna may act as both concomitantly. tumour-related mirna are more stable to processing than other molecules, making them optimal tumour biomarkers [127] . studies utilising mirna as non-invasive biomarkers for cancer detection are summarised in table 5 . colorectal cancer ng et al. described the significant overexpression of mir-17-3p and mir-92 in plasma from crc patients versus control subjects. mir-92 marker specifically differentiates crc from gastric cancer markers, making it to more sensitive crc marker [152] . similarly, a meta-analysis observed an increase in mir-17 in plasma/serum/faecal levels of crc patients, with 68% specificity [156] . mir-92a together with mir-21 was highly increased in serum samples, possessing prognostic value in crc patients [156] . further, results from the analysis of crc patient plasma revealed significant upregulation of a mirna panel (mir-15b, mir-18a, mir-19a, mir-19b, mir-29a and mir-335) depicting different mirna expression patterns amongst crc patients and healthy subjects [91] . further experiments validated these results, with 91% sensitivity and 90% specificity for crc and advanced adenoma (aa) detection and prognosis [153] . plasma mir-18 [154] and serum mir-19a [155, 160] have also been described to be significantly increased in crc patients in comparison with healthy controls. mir-29a has also shown an important role as a potential biomarker for crc detection. furthermore, mir-29a combined with mir-92a are capable to distinguish advanced crc from healthy subjects with 83% sensitivity and 84.7% specificity [158] . mir-21 has been extensively reported in multiple cancers as promoting proliferation and tumour growth, being one of the most relevant diagnostic mirna oncogenes in tumour onset [189] . a study testing 380 mirna described 19 dysregulated mirna in crc patient plasma samples. mir-21 upregulation discriminated crc patients from healthy subjects with a sensitivity and specificity of 90% [159] . furthermore, two independent studies revealed upregulation of mir-21 levels in crc patients compared with controls even years prior to the clinical manifestation of the disease [160, 161] . exosomal mirnas, although still insufficiently investigated, are increasingly applied as biomarkers for cancer detection featuring high specificity. for instance, increased serum levels of exosomal mir-19a and mir-92a in crc patients against controls have been detected [162] . similarly, upregulation of serum exosomal mir-21 and mir-23 amongst others, was described in crc patients [163] . in breast tumour studies, many differentially expressed mirna have been detected in breast cancer patients compared with healthy women. mir-1, mir-92a, mir-133a and mir-133b have been described as some of the most prevalent upregulated biomarkers in breast cancer samples [164] . another study revealed mir-182 serum levels to be increased in patients with breast cancer versus controls. furthermore, serum mir-182 levels were significantly lower in oestrogen receptor (er)-and progesterone receptor (pr)-positive breast cancer patients than those in er-and prnegative patients, demonstrating their clinical utility for breast cancer diagnosis [165] . additionally, 4 upregulated plasma mirna (mir-148b, mir-376c, mir-409-3p and mir-801) managed to discriminate breast cancer patients from controls [166] . mir-34a, mir-93 and mir-373 serum levels were distinguishable between m0 breast cancer patients on one hand and healthy subjects on the other hand, whilst mir-17 and mir-155 differentiated m0 from m1 patients [167] . further studies described mir-21 and mir-146a as increased in plasma levels, therefore, distinguishing breast cancer patients from healthy controls [168] . similarly, another study revealed mir-21 increased serum levels, which together with mir-10b, mir-125b, mir-145, mir-155 mir-191 and mir-382 are indicative for breast cancer occurrence [169] . moreover, two independent studies have described mir-21 increase to be of importance to discriminate breast cancer patients from healthy women [170, 171] . increased mir-21 concentrations corresponded with visceral metastasis [171] . mir-92 decreased levels along with elevated mir-21 were positively associated with lymph node detection and tumour size [170] . a microarray panel study analysing 1100 mirnas found 59 differentially expressed mirna in whole blood from early stage breast cancer patients against healthy individuals, from which 13 were up-regulated and 46 were downregulated [172] . looking for differences specific for breast cancer, 8 up-regulated and one downregulated plasma mirna were discovered: mir-16, mir-21 and mir-451 were significantly increased and mir-145 significantly reduced in breast cancer patients [173] . lung cancer to date, lung carcinogenesis molecular signature has been mainly monitored through mrna systematic analysis along with detection of protein expression levels [190] . however, mirna expression pattern analysis may portray novel diagnostic and prognostic tools for predictive and early lung cancer detection [191] . indeed, a study assessing mirna expression in early-stage nsclc serum samples revealed significantly increased mir-1254 and mir-574-5p levels, allowing for discrimination of nsclc patients from controls with a 77% and 82% specificity and sensitivity, respectively, and in the validation cohort with a 71% specificity and 73% sensitivity [174] . furthermore, 10 serum mirna (mir-20a, mir-24, mir-25, mir-145, mir-152, mir-199a-5p, mir-221, mir-222, mir-223 and mir-320) were detected to be differentially expressed in nsclc serum patient samples compared with controls. this specific mirna profiling was able to detect nslcl 33 months prior to the clinical manifestation of the disease [175] . 34-mirna signature model was created to detect early-stage nsclc within a population of highrisk asymptomatic subjects with an 80% accuracy [176] . in another study, mir-21 increased levels positively correlated with lymph node and tumour-node metastases in nsclc patients; shorter 3-year overall survival compared with patients with low levels of mir-21 expression was demonstrated [177] . similarly, mir-21 as well as mir-126, mir-210 and mir-486-5p were detected as a potential nsclc diagnostic panel, portraying 86.2% of sensitivity and 96.6% specificity [181] . contrarily, another study found that mir-21 along with mir-15b, mir-17, mir-28-3p, mir-106a, mir-126, mir-142-3p, mir-148a, mir-197, mir-221 and mir-486-5p were decreased in poor prognosis lung cancer cases [180] . nsclc serum patient study described increased levels of mir-30d and mir-486 together with decreased levels of mir-1 and mir-499 as correlated positively with poor nsclc prognosis [178] . plasma mirna analysis revealed mir-155, mir-182 and mir-197 levels to be considerably higher in lung cancer patients than in controls with a sensitivity of 81.33% and a specificity of 86.76%. higher pattern values were detected in patients with metastasis than in those without [179] . furthermore, mir-21, mir-126, mir-155 and mir-223 (84% specificity and 83% sensitivity) have arisen as a potential biomarker signature for lung cancer detection [182] . altogether, these studies suggest that corresponding mirna panels (but individual mirnas) have a predictive power for lung cancer detection. breast mir-1, mir-92a, mir-133a and mir-133b (upregulated) serum n = 132 breast cancer patients and n = 101 healthy subjects (control) [164] breast mir-182 serum n = 46 breast cancer patients and n = 58 healthy subjects (control) [165] breast mir-148b, mir-376c, mir-409-3p and mir-801 plasma n = 127 sporadic breast cancer cases and n = 80 healthy subjects (control) [166] breast mir-34a, mir-93 and mir-373 serum n = 120 patients with primary breast cancer (m0), n = 32 patients with overt metastasis (m1) and n = 40 healthy subjects (control) [167] breast mir-21 and mir-146a plasma n = 14 breast cancer patients and n = 8 healthy subjects (control) [168] breast prostate cancer prostate cancer (pc) diagnosis, monitoring and prognosis are widely based on the androgen-regulated genes and prostate-specific antigen (psa) [192] . in recent years, mirna have been described to impact cancer features by either promoting (oncogenic mirna) or suppressing (suppressive mirna) tumour development and disease progression [193] . pc often presents with a deregulation of mirna that may operate as oncogenes or tumour suppressors [194] . indeed, increased mir-141 levels were shown in pc serum samples [184] . increased expression of mir-18a was strongly correlated with promotion of pc, acting as an oncogenic mirna allowing discrimination between pc and benign prostatic hyperplasia (bph) [183] . moreover, mir 182-5p and mir-375-3p blood levels were detected as non-invasive screening signature and potential prognostic biomarker for pc development [185] . there is an accumulated evidence of numerous mirna tested in prostate cancer tissue samples acting as tumour suppressors [195, 196] . erg is able to bind to mir-200b/ a/429 assisting transcription in pc cells in tumour tissues [196] . moreover, mir-135a-1 was described to act as a potential tumour suppressor in metastatic pc by aiming at egfr [195] . another study revealed serum circulating mir-372 involvement in the progression of human pc by aiming p65mediated nf-κb/mmp-9/psa signalling pathway. thus, targeting mir-372/p65 interplay or interceding in mir-372 expression may present a valuable tool for diagnosis and treatment of pc patients [186] . however, studies addressing mirna panels pc specificity for example against prostate inflammation are needed. association between diabetes mellitus and carcinogenesis: diagnostic and therapeutic potential of cell-free nucleic acids diabetes mellitus gathers several metabolic diseases characterised by a chronic state of hyperglycemia. it can result in a deficiency in secretion of insulin, lack of insulin effect or both simultaneously. different types of diabetes exist, namely prostate mir-372 serum n = 20 serum samples from prostate cancer patients versus n = 20 healthy subjects (control) [186] type 1, type 2 and gestational diabetes, that differ in genetics and aetiology [197] . type 1 diabetes (t1d) is an autoimmune disorder characterised by hyperglycemia and β cell destruction [198] , whereas type 2 diabetes (t2d) is considered a metabolic syndrome. published epidemiological evidences portray a correlation between diabetes and cancer risk [199] . there are several potential risk factors common to both diseases, such as age, gender, diet, physical activity and obesity, amongst others [200] . diabetic patients present increased blood glucose levels, along with advanced glycation endproducts (age) that eventually leads to higher levels of dna damage [201] . studies have described age capability to cause dna strand breaks in colon and liver cells, as well as in murine podocytes. metabolic stress, mitochondrial impairments and insufficient dna repair increase risk of all-site carcinogenesis and cancer progression in diabetic patients [38, 201] . for example, correspondence between diabetes and crc has been described in numerous studies [202] . in fact, a study revealed a 5-year decreased overall crc, colon and rectal cancer survival (18, 19 and 16%, respectively) in patients with diabetes [203] . another study showed an increased risk in diabetic women of developing crc than men [204] . in women a direct risk by diabetes for breast cancer development has been described. a meta-analysis showed an increased cancer risk in diabetic women versus non-diabetic individuals [205] . a potential link between diabetes and breast cancer is promoted by oestrogen levels [206, 207] . in a lung cancer study contrasting lung cancer patients with and without diabetic history, diabetes was not a detrimental factor for lung cancer survival [208] . prostate cancer and diabetes studies have resulted in dissimilar outcomes. for one, a meta-analysis study revealed diabetic men to have decreased risk of developing prostate cancer [209] . another study described an increase in 29% in prostate cancer-related mortality in diabetic patients compared with non-diabetic subjects [210] . obviously, a detailed patient stratification by individualised patient profiling is essential to bring more consensus in the data interpretation that allows for a disease prediction and of high quality personalised services to the patient [9] . anti-diabetic drugs are known to decrease diabetes pathophysiological factors (high blood glucose and age), however, drugs such as metformin may also reduce risk of cancer in diabetic patients. in fact, studies have postulated anti-oxidant properties of anti-diabetic drugs and renin-angiotensin system inhibitors to potentially reduce cancer risk [211, 212] . diagnostic and therapeutic potential of cell-free nucleic acids in diabetes determining differentially expressed mirna or differentially methylated β cell derived dna might better identify t1d cohorts, as mirna are known to be imperative in t1d pathogenesis and regulating β cell function [213] . the use of proinsulin/ c-peptide (pi/c) ratios may support identification of β cell destruction in subjects prior to the development of t1d, serving as a non-invasive marker of β cell malfunction [214] . mirna-375 has been reported as being one of the most abundantly expressed mirna in β cells. in fact, mice lacking mir-375 appeared to have decreased β cell mass and increased glucagon secretion, resulting in a hyperglycemic state [215] . a similar study portrayed an overexpression of mir-375 in primary mouse islets [216] . consequently, mir-375 was tested as a potential biomarker for diabetes. in fact, increased mir-375 was detected in mice prior to hyperglycemia onset [217] . mirna-375 plasma levels were elevated in patients at 7 days post islet transplantation [218] . serum mirna sequencing analysis has identified mir-52, mir-24, mir-25, mir-26a, mir-27a, mir-27b mir-29a, mir-30a-5p, mir-148a, mir-181a, mir-200a and mir-210 as differentially expressed in t1d patients [219] . further studies have tested mirna patterns in immune cells from t1d patients, revealing an increased expression of mir-326 in lymphocytes from t1d subjects [220] . another study determined decreased expression of mir-146 in pbmc from t1d patients against non-diabetic controls [221] . evaluation of increased unmethylated insulin dna in circulation is a key to detect evolution of t1d resulting from βcell death [222] . two independent studies revealed higher unmethylated to methylated insulin dna ratios versus nondiabetic controls [223] and higher circulating levels of both methylated and unmethylated insulin dna in early onset t1d patients [224] . similarly, plasma cell-free dna levels from new onset t1d and allogeneic islet transplantation subjects were higher than in controls [225, 226] . furthermore, t2d patient's serum was tested for specific mirna profiles: t2d were compared with obese patients and healthy controls. combined mir-138 and mir-503 patterns enabled to discriminate between diabetic and obese diabetic patients. further, using mir-15b in combination with mir-138 and mir-376a may help to distinguish between t2d and obesity. this evidence makes serum mirna profiling to a potential t2d predictive tool [227] . furthermore, a study which investigated plasma mirna profiles in t2d patients revealed diminishing plasma levels of 10 mirna and a slight increase of mir-28-3p. in fact, analysis of mir-15a, mir-28-3p, mir-126, mir-223 and mir-320 represent a suitable t2dm signature array [228] . ischemic stroke [85] pathology-specific versus common cell-free nucleic acid patterns imbalanced stress-and ischemia-related disorders, diabetes and cancer share several risk factors such as toxic environment, suboptimal life-style and dietary habits, specific phenotypes, vasospasm, accelerated ageing and abnormal body weight (both underweight and obesity), amongst others [9, 10, 13, 113, [229] [230] [231] [232] . to this end, diabetes mellitus has been demonstrated as a prominent example of cancer risk factor [200] . unfortunately, in many cases, studies do assess potential biomarkers out of context of collateral pathologies and potentially related health conditions that has been strongly criticised in the literature [233] . those deficits should be compensated via well designed further studies, on one hand to indicate common origin and molecular pathways involved in several and collateral pathologies [234] . on the other hand, pathology-specific patterns are of great value for predictive diagnostic purposes, targeted prevention and cost-effective personalisation of medical services [6, 235] . table 6 provides examples for pathology-specific and table 7 for common cfna panels in health conditions and disorders which the current paper has referred to. [80, [84] [85] [86] , cfdna and mtdna could act as promising diagnostic and prognostic biomarkers of ischemic stroke [77, [81] [82] [83] . the mir-19a dysregulation has been described in numerous studies related to crc cancer patients compared with controls [91, 153, 155, 160, 162] . moreover, mir-335 was also detected in crc tumours by several different groups [91, 153] . breast cancer specific biomarkers detected are mir-34a, mir-382, mir-146a and mir-451 [167] [168] [169] 173] . patterns of mir-197 [179, 180] and mir-486-5p [178, 180, 181] have been analysed as pathology-specific biomarkers in lung cancer patients. moreover, prostate cancer specific biomarkers are mir-135a-1, mir-141, and mir-372, amongst others [184, 186, 195] . one of the most prominent diabetes mellitus biomarker is mir-375 [215] [216] [217] along with further biomarkers described as specific for diabetes detection such as the panel of mir-138, mir-181a, mir-326 and mir-376a, amongst others [219, 220, 227] . table 6 summarises pathology-specific cfna panels. comprehensive analysis demonstrated common cfna panels amongst different diseases. for instance, mir-21 has been described to be present in crc, breast and lung cancers. mir-21mir-320 stress-lung cancer-diabetes [48, 175, 228] 5p and mir-223 are related to both ischemic stroke and crc cancer. furthermore, mir-223 has been described for lung cancer, ischemic stroke, diabetes and stress-related diseases. lung cancer shares several mirna markers with stress-related disorders (mir-28-3p, mir-182, mir-223 and mir-320) and physical activity (mir-126, mir-221 and mir-222). mir-16 and mir-182 are present in both breast cancer and stress-related diseases. moreover, mir-15b, mir-24, mir-25, mir-28-3p, mir-126, mir-148a and mir-320 were present in lung cancer and diabetic patients. dysregulation of mir-21, mir-145 and mir-155 have been found in both breast and lung cancer patients. whereas, mir-15b, mir-17and mir-20a are common markers in crc and lung cancers. table 7 summarises common cfna panels. further studies addressing interrelations amongst human disorders and shared cfnas signature are essential. what is known about cfnas signature utility in covid-19 management? many research teams around the world are intensively working on prediction of the covid-19 epidemics, protective measures to populations, therapeutic and vaccination issues. it has been clearly demonstrated that lack of specific diagnostic laboratory tools may lead to incorrect political decisions causing either unnecessary overprotection of the population that is risky for a long-term economic recession, or underprotection of the population leading to a post-containment pandemic rebound [237, 238] . blood parameters are highly indicative for the patient stratification, disease cause and individual outcomes [239] . patients demonstrating severe course of covid-19-related disease suffer from cytokine storms and multiorgan failure [240] ; however, the underlying mechanisms still remain uncertain. available information demonstrates that profuse innate immune responses aggravate individual outcomes [241] . viral infections have been described to prompt cellular necrosis, which amplifies anti-viral immune responses releasing damage associated molecular patterns (damps) [242] . severely affected cells and tissues intrinsically secrete cfnas such as mitochondrial dna (mt-dna) [243] . it has been demonstrated that covid-19 patients with increased levels of mt-dna are at elevated death risk, necessity of icu care and intubation. consequently, cell-free mt-dna is a potential biomarker for individualised survival status prediction [243] . lb and individualised profiling of biomarker patterns presented in body fluids represent a revolutionary approach in the work-frame of 3p medicine. in the last years, cfnas signature attracted a lot of attention for diagnostic and treatment purposes. altered profiles of cfnas have been detected under both physiological and pathological conditions. although oncological research is particularly advanced implementing cfnas for diagnostic and treatment purposes, independently of the application area, the main goal remains the same, namely to look for pathology-specific biomarker patterns as well as for patterns clearly indicating associated risks, for example, in vasospastic individuals who are a prominent example of patients predisposed to an increased stress sensitivity, neuro/degenerative disorders and/or aggressive metastasing cancers as discussed above. this article highlights the involvement of cfnas in local and systemic processes dealing with the question, whether specific patterns of cfnas in blood, their detection, quantity and quality (such as methylation status) might be instrumental to predict a disease development/progression and could be further utilised for accompanying diagnostics, targeted prevention, creation of individualised therapy algorithms, therapy monitoring and prognosis. presented considerations conform with principles of 3p medicine [244] and can be implemented for improving individual outcomes and cost-efficacy of medical services provided to the population. author contributions og and pk have coordinated the project and created the concepts of the study. atc, ms, lk, al and og have performed literature search and drafted the manuscript. atc, ms, fag, pk and og have elaborated on the final version of the manuscript. all authors have read and approved the final version. funding open access funding enabled and organized by projekt deal. conflict of interest the authors declare that they have no conflict of interest. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and 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with prostate cancer: a potential novel non-invasive biomarker circulating micrornas as stable blood-based markers for cancer detection circulating micrornas as biomarkers for prostate cancer detection and metastasis development prediction microrna-372 suppresses migration and invasion by targeting p65 in human prostate cancer cells micrornas in human diseases: from cancer to cardiovascular disease microrna function in cancer: oncogene or a tumor suppressor? oncomirs-micrornas with a role in cancer genomic and proteomic profiling of lung cancers: lung cancer classification in the age of targeted therapy micrornas as therapeutic targets in lung disease: prospects and challenges. chronic obstr pulm dis prostate cancer screening with prostate-specific antigen testing: more answers or more confusion? microrna signatures in human cancers micrornas in prostate cancer: an overview circulating tumor cells, dna, and mrna: potential for clinical utility in patients with melanoma the tumor suppressive mir-200b subfamily is an erg target gene in human prostate tumors diabetes mellitus: the epidemic of the century circulating unmethylated insulin dna as a potential non-invasive biomarker of beta cell death in type 1 diabetes: a review and future prospect diabetes and cancer: a comprehensive review diabetes and cancer: a consensus report. diabetes care. 1674-1685 evidence for dna damage as a biological link between diabetes and cancer the relationship between diabetes and colorectal cancer prognosis: a meta-analysis based on the cohort studies association of type 2 diabetes mellitus and the risk of colorectal cancer: a meta-analysis and systematic review diabetes increases the risk of breast cancer: a meta-analysis postmenopausal serum androgens, oestrogens and breast cancer risk: the european prospective investigation into cancer and nutrition diabetes mellitus and risk of breast cancer: a meta-analysis patient outcomes from lung cancer and diabetes mellitus: a matched case-control study a meta-analysis of diabetes mellitus and the risk of prostate cancer diabetes and mortality in patients with prostate cancer: a meta-analysis diabetes and cancer: the mechanistic implications of epidemiological analyses from the hong kong diabetes registry additive effects of blood glucose lowering drugs, statins and renin-angiotensin system blockers on all-site cancer risk in patients with type 2 diabetes circulating micrornas as novel biomarkers for diabetes mellitus islet β-cell endoplasmic reticulum stress precedes the onset of type 1 diabetes in the nonobese diabetic mouse model mir-375 maintains normal pancreatic alpha-and beta-cell mass a pancreatic islet-specific microrna regulates insulin secretion circulating mir-375 as a biomarker of β-cell death and diabetes in mice evaluation of microrna375 as a novel biomarker for graft damage in clinical islet transplantation circulating levels of microrna from children with newly diagnosed type 1 diabetes and healthy controls: evidence that mir-25 associates to residual beta-cell function and glycaemic control during disease progression increased expression of microrna mir-326 in type 1 diabetic patients with ongoing islet autoimmunity decreased mir-146 expression in peripheral blood mononuclear cells is correlated with ongoing islet autoimmunity in type 1 diabetes patients 1mir-146 insulin gene expression is regulated by dna methylation β cell death and dysfunction during type 1 diabetes development in at-risk individuals elevations in circulating methylated and unmethylated preproinsulin dna in new-onset type 1 diabetes detection of β cell death in diabetes using differentially methylated circulating dna identification of tissue-specific cell death using methylation patterns of circulating dna serum circulating microrna profiling for identification of potential type 2 diabetes and obesity biomarkers plasma microrna profiling reveals loss of endothelial mir-126 and other micrornas in type 2 diabetes pregnancy-associated breast cancer: the risky status quo and new concepts of predictive medicine diabetes care in figures: current pitfalls and future scenario vaginal dryness: individualised patient profiles, risks and mitigating measures flavonoids against the warburg phenotype-concepts of predictive, preventive and personalised medicine to cut the gordian knot of cancer cell metabolism inappropriate modeling of chronic and complex disorders: how to reconsider the approach in the context of predictive, preventive and personalized medicine, and translational medicine common origin but individual outcomes: time for new guidelines in personalized healthcare preventive, predictive, and personalized medicine for effective and affordable cancer care hsa-mir-135a-1 inhibits prostate cancer cell growth and migration by targeting egfr covid-19 pandemic by the "realtime" monitoring: the tunisian case and lessons for global epidemics in the context of 3pm strategies covid-19 what have we learned? the rise of social machines and connected devices in pandemic management following the concepts of predictive, preventive and personalized medicine early decrease in blood platelet count is associated with poor prognosis in covid-19 patients-indications for predictive, preventive, and personalized medical approach sars-cov-2 pandemic: clinical picture of covid-19 and implications for research immunology of covid-19: current state of the science necroptosis in anti-viral inflammation circulating mitochondrial dna is an early indicator of severe illness and mortality from covid-19 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-006860-a3b8hyyr authors: nan title: 40th annual meeting of the gth (gesellschaft für thromboseund hämostaseforschung) date: 1996 journal: ann hematol doi: 10.1007/bf00641048 sha: doc_id: 6860 cord_uid: a3b8hyyr nan the variable molecular weight (mw) of vwf is due to differences in the number of subunits comprising the protein. it is assumed that endothelial cells secrete large polymeric forms of vwf and that smaller species arise from proteolytic cleavage. vwf has two main properties: it stabilizes factor viii protecting it from inactivation by activated protein c or factor xa, and it mediates platelet adhesion to subendothelium of the damaged blood vessel wall. each vwf subunit contains binding sites for collagen and for platelet giycoproteins gp ib and gp iib/i~a. multiple interactions of the multivalent vwf lead to extremely strong binding of platelets to subendothelial surface, that is capable of resisting high wall shear rate in the circulating blood. only the largest multimers are hemostatically active. lack of the largest vwf multimers was observed in patients with yon wiuebrand disease type 2a. unusually large molecular forms of vwf were found in patients with thrombotic thrombocytopenlc purpura. proteolytic enzyme(s) may be involved in the physiologic regulation of the polymeric size of vwf and thus play an important role in the pathogenesis of vwf abnormalities in some patients with congenital or acquired disorders of hemostasis. we have purified (-10,000-fold) from human plasma a vwf degrading proteas¢ using affinity chromatography and gel filtration. the proteolytic activity was associated with a high mw protein (mr -300 kd). vwf was resistant against the protease in a physiologic buffer but became degraded at low salt concentration or in the presence of 1 m urea. proteolytic activity had a ph optimum at g-9 and was not inhibited by serine protease inhibitors or sulfl~ydryl reagents. inhibition by chelating agents was best reversed by barium ions, the observed properties of the vwf degrading enzyme differ from those of all hitherto described pretenses. analysis of cleaved vwf showed that the peptide bond 842tyr-g43met had been cleaved -the same bond that has been proposed to be cleaved in rive. the endothelium releases the vasodilator nitric oxide (no) and the vasoconstrictor endothelin (et)-i. no is formed from l-arginine via the activity of constitutive nitric oxide synthase (cnos or enos). an inducible form of nos (inos) is activated by cytokines. no activates guanylyl cyclase in vascular smooth muscle and platelets, leading to the formation of cgmp which induces relaxation or platelet inhibition, respectively. in vessels, no is responsible for endothelium-dependent relaxations; in vivo it exerts a vasodilator tone which can be enhanced by shear forces and receptor-operated agonists such as acetylcholine, bradykinin, thrombin, atp and adp. infusion of no-inhibitors in vivo leads to vasoconstriction and increases in blood pressure and oral administration to hypertension in the rat. within the endothelium, no inhibits et gene expression and release of the peptide via cgmp. hence, no-induced hypertension is associated with increased plasma et levels. et, a 21-amino acid peptide, has potent vasoconstrictor properties via eta-and in part etb-raceptors on vascular smooth muscle. in endothelial cells, et activates etb-receptors linked to no and prostacyclin formation. under basal conditions, little et is formed, but is increased by thrombin, angiotensin ii, arginine vasopressin, cytokines and ox-ldl. et antagonists have been developed and allow to study the effects of et in vivo. et and no most likely play an important role in disease states such as hypertension, atherosclerosis, coronary artery disease, heart failure, pulmonary hypertension and subarachnoid hemorrhage. clinical trials to further define their role in these disease states are now under way. in summary, the endothelium is an important regulator of vascular tone and structure in vitro and in vivo. in disease states, their interaction is imbalanced leading to enhanced vasoconstriction, thrombus formation and structural changes of the blood vessel wall. pharmacological tools aiming to inhibit those changes are now being developed. j.m. harlan, r.k. w/nn, s. sharar, and n. vedder universit3, of washington, seattle, washington ischemia-reperfusion injury has been implicated in the pathogenesis of a wide variety of efinical disorders. [n preclinical models, tissue damage .clearly occurs during ischemia, but, parado.,dcally, may be exacerbated during reperfusion. this reperfusion injury appears to involve activation of the intlammato~, cascade with generation of complement 'components, lipoxygenase products, and chemokines as proximal mediators and neutrophils as final effectors of vascular and tissue damage. we have examined the role of leukocyte adhesion in reperfusion injury in two models -the rabbit ear as a model of isolated organ injury and hemorrhagic shock and resuscitation in the rabbit and primate as a model of traumatic shock and multiple organ failure. data regarding the efficacy, timing, and safety of leukocyte adhesion blockade using selectin-or integrindirected reagents in these models w/ll be presented. the current status of anti-adhesion therapy in other pre-clinical models and early clinical trials will be re~4ewed. an amidolytic assay for the determination of activated protein c (apc)-resistant factor va (fva) has been developed. this assay measures the cofactor activity of fva in diluted plasma samples via the rate of thrombin formation. the apc response is calculated from two fv determinations: one performed in the presence (apc-fv) and one in the absence of recombinant apc. the apc-fv activity is expressed as percentage of the initial fv activity and indicates the sensitivity of fva to apc. normal ranges were established by analysing plasma samples of 100 healthy individuals and an apc-fv activity above 60% was found to be indicative for apc-resistance (apc-r). in a control group of 34 patients the apc-r assay gave abnormal results in 15 patients. dna analysis confirmed heterozygous fv r506q mutation in all 15 patients and confirmed the non-carrier status in all of the 19 patients yielding normal results. an aptt-based apc-r assay performed on the same group of patients showed abnormal results in two of the non-carrier patients. one of these patients was diagnosed as positive for lupus anticoagulant, whereas the reason for the wrong positive result in the second patient remains unclear. eleven patients were analyzed before start of oral anticoagulation and during oral anticoagulant treatment. comparison of the assay results demonstrate a correlation of 96% indicating that the assay is independent of the activities of vitamin k-dependent clotting factors. the apc-r amidolytic assays allows specific and sensitive detection of fva-resistant to apc. the assay is performable in plasma samples of all persons in whom the diagnosis of apc-r may be indicated. in patients treated with oral anticoagulants or showing other clotting abnormalities affecting the aptt the apc-r amidolytic assay is helpful to establish the diagnosis of apc-r. dept of pediatrics, university hospitals kiel and mtinster, germany resistance to activated protein c (apcr), in the majority of cases associated with the arg 506 gin point mutation in the factor v gene is present in more than 50 % of patients < 60 years of age with unexplained thrombophilia. to determine to what exteut this relativdy common gene mutation affects the risk of thromboembolie events in infants and children, its occurrence was investigated in a population of children with unexplained venous or arterial thromboernbotism: thrombosis of the central nervous system (cns, n=4), vena portae (n=4), deep vein thrombosis (n=4), vena caval occlusion (n=4), neonatal renal venous thrombosis (rvi'; n--3), neonatal stroke (n=14), stroke (n=3), arteria femoralis ocdusion (n=2). four ont of these 38 patients showed a positive history of unexplained familial thrombophilia. apcr was measured in an activated thromboplastin time (afit) according to dahlbtick. the results were expressed as apc-ratios: clotting time obtained using the apc/caci2 -solution divided by dotting time obtained with cac12. concerning the special properties of the childhood hemostatic system, infants and children with apcr < 2 were considered to be apc-resistent only when the results were confirmed in a 1: l 1 dilution with factor v deficient plasma (instrumentation laboratory munich, germany). plasma of 268 healthy children served as controls. the arg 506 gin mutation of the factor v gene was assayed by amplification of the dna samples by pcr followed by digestion of the amplified products with the restriction enzyme mul i. results were confirmed by sscp -analysis or by direct sequencing of dna from patients with apcr. consistent with the ap'it based method 10 out of 19 children with venous (v) thrombosis and eight out of 19 patients with arterial (a) vascular insults showed the common factor v mutation. additional coagulation defects (autithrombin, protein c type i, enhanced antiphospbolipid igg, enhanced lipoprotein (a)) were found in 30% (v) and 28% (a). furthermore, we diagnosed exogenlc reasons (septicemia, postpastal asphyxia, fetopathia diabetica, central line and steroid/asparaginase administration) in six out of 10 (v) and three out of 8 (a) children with thrombosis and apcr. all four patients with a positive family history of thrombophilia (mothers only !) showed the common factor v mutation arg 506 gin. in the control group the prevalence of apcr was 5.1%. the high incidence of additional exengenic factors in children with apcr confirm literature data of previously described inherited coagulation disorders during infancy and childhood: an acquired risk of thromboembolic disorders masks the coagulation deficiency in the majority of patients with an inherited prethrombotic state. furthermore, the incidence of 42 % apc resistant children with arterial insults in this study challenge the view that apcr is associated with venous but not with arterial thrombo-st8. 12 activated protein c resistance and plasminogen deficiency in a family with thrombophilia m, zttger 1 , f. demarmels biasiutti 1, ch. mannhalter 2, m. furlan 1 , b.~e 1 1httmatologisches zentrallabor der universit~t, inselspital, ch-3010 bern 2klinisches institut fur medizinische und ehemische labordiagnostik, universit~t wien, a-1090 wien several hereditary defects of the proteins regulating blood coagulation have been associated with familial thrombophilia. since the recent discovery of activated protein c (apc) resistance due to the factor v rf06q mutation as a highly prevalent hereditary risk factor for venous thromboembolism (tel, evidence is accumulating that familial thrombophilia may be due to a combination of genetic defects. thus, protein c-or protein s-deficient patients having suffered from te seem to be more likely to carry the factor v r506q mutation than expected from its allelic frequency in the population. we report a family (see figure) in which plasminogen deficiency (0.60 u/ml) had been found in the propositus having had twice postoperative deep vein thrombosis (dvt) at ages of 29 and 31 yrs, respectively, as well as in 5 family members (0.53-0.69 u/ml). out of these 5 plasminogen deficient individuals, only the propositus' daughter had suffered from recurrent dvt at age <20yrs. reinvestigation of this family in 1995 showed factor v r506q mutation in the propositus, his daughter, an asymptomatic sister and a brother with postoperative pulmonary embolism (pe). his father had had postoperative pe; he is deceased and could not be examined. ~, [] plasminogen deficiency ~ ,rll factor v rf06q mutation /~ propositus ~¢ bistory of dvt and/or pe e superficial phlebitis j2~,,~ not investigated "1" deceased even though this family is small for establishing unequivocal association of te with known defects, the two most severely affected individuals with recurrent te at ages <30yrs had combined plasminogen deficiency and apc resistance whereas those with isolated plasminogen deficiency were asymptomatic. these data support the concept of multigenie interactions leading to familial thrombophilia. resistance to activated protein c (apc resistance) is the most common dsk factor for venous thrombosis (vt). in most cases apc resistance is caused by a single point mutation at position arg 506 in the factor v gene (factor v leiden). while ample data in hetarozygous patients have been published, reports in homozygous patients are limited. we studied 29 patients (12 males [m] , 17 females it]) in whom a homozygous mutation had been verified by dna analysis. the median age at the time of the study was 38.8 years (y) (range 9-83 y). twenty-five patients had experienced vt (10 m, 15 f). four patients were discovered dunng family studies and were asymptomatic, three were children (between 9 and 13 y) and one patient was a 62 y old man. in males the first thrombosis occurred at a median age of 38 y (range 21-82 y), in females this was at a significantly younger median age of 26 y (range 17-49 y). twelve of the 15 symptomatic females had taken oral contraceptives (oc, estregen content 0.02-0.ling) for 6 to 150 months (median 71 m) pdor to thrombosis. in 2 women vt occurred during pregnancy, in 1 female it was precipitated by hormone replacement therapy. in contrast, in 8,<10 males the thrombosis happened spontaneously, in 2 males it followed surgery. the sites of thrombosis were dvt in 9 males and 14 females, dvt and pulmonary embolism (pe) in 4 females and 1 male, dvt and caval vein thrombosis in 1 female and superficial thrombophlebitis in 4 males and 1 female. eight females had at least one pregnancy, in total 11 children and 5 abortions. two had thrombotic events during pregnancy and 2 after delivery. all homozygous patients showed apc ratios between 1.12 and 1.68 (mean 1.39 + 0.19). conclusion: patients with homozygous fv leiden have similar clinical symptoms as patients with deficiencies of antithrombin-, protein c or protein s deficiency. however, in contrast to these defects a very high dsk dudng oral contraceptive medication leading to an ealier manifestation in females can be observed. several synthetic (efegatran, argatroban, inogatran and napsagatran) and recombinant (hirudin, peg-hirudin and hirulog) antithrombin agents are in different stages of nlinical development for cardiovascular and thrombotic indications. while the specificity of these agents for thrombin is a concern, little has been done to study the effects of these agents on other serine proteases involved in coagulation and fibrinolytic processes. fihrinolytic compromise by site directed thrombin inhibitors has been reported recently (thromb res 74(3): 193-205, 1994) . while these agents have been shown to inhibit plasmin and related enzymes, little or no informatienentheir effects onthe generation and functionality of apc is available. sinceapc plays an increasingly important role both as an antienagulant enzyme, by inhibiting factors v and viii, end a pro-fibrinelytic enzyme, by stimulating the release of t-pa fi'om endothelial sites, an inhibition of apc may result in both pro-coagulant state and fibrinolytic deficit. representative thrombin inhibitors (dup 714 -a prototype boronic acid peptide derivative, efega~an, argatroban, hirulog, hirudin and feg-hirudin) have been compared for their ability to inhibit apc (american red cross). these bionhemically defined studies in which the remaining activity of apc after incubation with a thrombin inhibitor was determined speclrophotometrically with a ehromogenic substrate (s-2288, pharmacia, franklin, oh) , demonstrated that dup 714 and efegatran inhibit apc in a coneentrafinn dependent manner (ic~0=0.75 and 8,4 gm resp~tively), hirnlog inhibits apc weakly (60 p2¢i produced only 25% inhibition), while argatroban and ~ have no anti-apc activities, while hirulog, hirudin and argatroban produced no direct enti-apc activities, it is conceivable that they may inhibit thrombomodnlin-bound thrombin and thus prevent activation of protein c, resulting in a functional apc deficit and failure to improve clinical outcomes despite higher dosage. while initially it was thought that sole targeting of thrombin will provide monospacifin anticoagulant agents devoid of some of the adverse effects observed with heparin, the recent clinical trials clearly suggest that thrombin is not the only determinant of thrombogenesis. furthermore, potent antithrombin agents such as hirudin, hirulog and peptides, indirectly inhibit the generation of apc, by compromising thrombomodulin-bound thrornhin and such agents as efegalran and dup 714 also produce direct apc inhibition. endogenous inhibition of formed ape by thrombin inhibitors may therefore compromise the feedback regulatory funetiens of apc and may lead to thrombotic amplification in fully enticoagulated patients. these studies warrant prcelinlcal assessment of thrombin inhibitors to evaluate their relative inhibitory effects on apc. poor anticoagulant response to activated protein c (apc resistance) causes a significant portion of deep vein thrombosis (dvt) whereas its association with coronary artery disease (cad) and myocardial infarction (md is still controversial. therefore, we investigated 346 recently hospitalised patients suffering from cad with or without previous mi. the cad was proven by coronary angiography. apc resistance was analysed by using the ap'l-i'-based assay coatest apc resistance (chromogenix). eleven patients showed an apc sensitivity index below 2.0 viewed as apc resistance. using pcr technology, the factor v mutation causing apc resistance 11691 g "-) a) has been shown in nine of these eleven patients. this represents 2.6 % (9/346), compared to 8,5 % found in healthy blood donors (8/94). one homozygous carrier (male, age 76) was identified (apc sensitivity index 1.4) who suffered from dvt at age 59. recent angiography demonstrated diffuse cad, no thrombotic events were reported in his family. in contrast, multiple thrombotic manifestations (dvt, mi, stroke) occurred in the relatives of four heterozygous patients. we conclude that the prevalence of apc resistance is rather low in patients with cad. nevertheless, the natural history of coronary manifestation of apc resistance seems to vary, probably depending on the presence and severity of cardiovascular risk factors. resitanee to activated protein c (apc resistance) is the most cormnon hereditary cause of thrombophilia and significantly linked to factor v leiden pcr based methods are used to identify the crucial point mutation in the factor v g(me. we designed primers in order to identify factor v leiden by allele-specific pcr amplification. amplification specificity for factor v was ensured by the 3'primer fv1001, located at the introng/intronl0 border of the g~ae. one sense and two antisense primers were used in ~vo separate primer mixes specific for factor v arts06 (wildtypo) or factor v otn506 (factor v leiden) yielding 235bp products each. in each pcr reaction a pair of primers amplifying a fragment of the human growth hormone gene was included, fimctioning as an internal positive amplification control (429bp pcrfragment). after an initial denaturation step 10/.tl samples (100rig genomic dna) were subjected to 10 two-temperature cycles fouowed by 20 threetemperature cycles. for visualisation 8p3 of the amplification product were run on a 2% agarose gel presmined with ethidittm bromide. the presence or absence of specific pcr amplification allowed defiu/te allele assignment without the need for any postamplifieation specificity step. the in~ernal positive control primers it~cate a sucessf-u/pcr amplification, allowing the assignment of homozygosity. in a prospective study 126 p~e.ients with tlaromboembolic events were analysed using this technique and enmpared with pcr -rflp according to bertina et al. the concordance between these techniques was 100 %. in 27 patients a heterozygous factor v ohas06 mutation was detected, whereas one pa6ent with recurrent thrombcembolism was homoz-ygous. no false-positive or false-negative results worn observed in the homozygous as well as hcterozygous samples. in addition in 15 samples identified to carry the point mutation by al/ele-specifin pcr anxplification automatic :~equencing confirmed the heterozygous or homozygous point mutation. due to its time-and cost-saving features allele-specific amplification should be considered for screening of factor v leiden. background: an initial intravenous course of tmfractionated heparin ~ljasted on the basis of the activated partial thromboplastin time is the currmt standard treatment for most patients with venous thrombosis. low-molecularweight hqmrin pre~a~tious can be administered subcutaneously, once or twice daily, without laboratory monitoring. we compared the relative effic.~y and safety of low-molecular weight heparin versus anfractionated heparin for the initial treatment of deep venous thrombosis. methods: english-language reports of randomized trials vtta'e identified th~ a medline search (1984 through 1995) and a complementary extensive manual search. reasons for exclusion from the analysis were no hepada dosage adjustments, the lack of um of obj~tive tests for deep venous thrombosis, dos~ranging studies that used higher doses of low-molecularweight heparin than are ctareatly in use, and the failure to provide blind endpoint ~sossmeat. we assessed the incidence of symptomatic recurrent vinous thromboembolic disease, the incidence of clinically ii~t bleeding and mortality. results: twelve of the 21 identified trials satisfied the predetermined criteria. the relative risk reductions for symptomatic thromboembolic complicatious, clinically ~t bleeding, and mortality varied firom 20.60% aad were all statistically significantly in favor of low-molecular-wedght hqtmrin. coadusions: low-mol~ular--weight hoparim administered subcutaneously in fixed doses adjusted for body weight aml without laboratory nmaitori~ =re more effective and safea" tlum adjn~_-dose standard h~. sauce low molec~dar weight hqmrim vary in o~apositiou =ad pharma~ogical im)fil~ the benefits of each ~ shodd ~tabllsbcd separately. unfraetionated hcparin (uh) and low molecular weight heparin (lmwh) are widely used for the prevention and treatment of thrombotic disorders. uh and lmwh induce platelet aggregation in vitro. rgd peptides compete with fibrinogm for the binding to the glycoprotein receptor (gp lib-ilia) of platelets and inhibit platelet aggregation. to inhibit the heparin-indueed platelet ~tion and prolong the half-life in blood of rgd peptides, we linked ac-rgdv-ssggs-ahx-yk eovalently to lmwh in a ratio 1:1. the peptide is composed of three regions: a. rgd-gives the specificity for the receptor gp lib-ilia; b..ssggs-ahx-is the spacer between carrier and ligand, which should facilitate the intnraetion between the conjugate and the gp lib-ills receptor; c. -yk arc functional antino acids for iodination (y) and for covalent attachment (k) to the cattier lmwh. the aggregation achieved with different concentrations of lmwh, lmwh-eonjugate and lmwh/rgd-peptide mixture in a ratio 1:1 was mea.~ared after 20 rain.; maximum aggregation after platelet activation with 10 pm adp was set equal to 100%. platelet aggregation in normal human plateletrich eitrated plasma (prp; 220 000/p.l) was induced by lmwh in a dose ~ndent manner. heparin can induce antbodies which interact with platelets and endothelial cells. this causes thrombocytopenia and thromboembolic complications. hitpatients do need effective parenteral anticoagulation. we treated 82 patients (30m,52fm), median age 61 years (18-84) with laboratory prooven hit (hipa-test) with recombinant (r-)hirudin. as these patients had been preseleeted by their immunological response during heparin treatment and the treatment duration of the study was longer than in any other study using thlrudin, all patient samples were investigated for anti-r-hirudin antibodies himdin antibodies were screened by a sandwich elisa using r-hirudin fixed to the solid phase as antigen. all plasma samples were screened for anti-hirudin antibodies of the igg class, but solar only a suset of samples for lge anti-r-himdin antibodies. 38 of 82 patients (46.3%) developed anti-hirudin antibodies of the igg class. anti-hirudin antibodies were not detectable not before 6 days of r-hirudin administration solar no ige anti-hirudin antibodies were found. none of the patients devdoped thromboeytopenia or allergic symptoms. however, in a subset of patients the anti-hirudin antibodies enhanced the anticoagulatory effect of r-hirudin. in 5 patients the hirudin dosage had to be decreased by 2-3 fold to maintain a stable aptt level, in 3 patients, despite stable r-hirudin maintenance dose the aptt increased to values >100 see.. during the study 4 patients with anti-hirudin antibodies had to be reexposed to a second course of r-hirudin for parenteral anticoaguhtion none of these patients developed any allergic reaction. in conclusion we found a high proportion of anti-hirudin antibodies in hatpatients treated with r-hirudin for more than 7 days. these antibodies seem to have minor clinical relevance in regard to allergic reactions. however, one has to consider that these antibodies may influence the pharmacokinetics of rhimdin and thereby enhance its antieoagulatory potency. therefore, aptt must be monitored closely in patients receiying r-hirudin for more than 5 days a major concern in the use of hirndin, the most potent and specific thrombin inhibitor, is the risk of bleeding associated with the potential effect of this drug on hemostasis, particularly when the antithrombotic therapy is combined with invasivo procedures, fibrinolytic treatment, or patient's predisposition to abnormal bleeding. thus, availability of an antagonist to hirudin would be essential for instant neutralization of the antithrombotie action. however, thueh a hirudin antagonist is unknown in nature. to prepare an antagonist to hirudin, a mutant derivative of human prothrombin, in which active site aspartate at position 419 is replaced by an asparagine, has been designed, expressed in recombinant chinese hamster ovary cells, and purified to homogeneity. d419n-prothrombin was converted to the related molecules d419n-meizothrombin and d419n-thrombin by limited proteolysis by e. carinatue venom and o. scvutellatus venom, respectively. both d419n-thrombin and d419nmeizothrombin exhibited no thrombin activity and titration resulted no detection of the active site. however, binding to solid phase immobilized hirudin and fluorescence studies confirmed that the binding to the most specific thrombin inhibitor, hirudin, was conserved in both proteins, hi vitro examinations showed that d419n-thrombin and d419nmeizothrombin bind to immobilized hirudin, neutralize hirudin as well as in the purified system and in human blood plasma and re-activate the thrombin-hirudin complex. animal model studies confirmed that d419nthrombin and d419n-meizothromi.,in act as hirudin antagonist in blood cireulatlon without detectable effects on the coagulation system. while i.v. injections of hirudin in mice resulted in an increase in partial thromboplastin time, thrombin time and anti-thrombin potential, additional injections of d419n-thrombin and d419n-meizothrombin resulted in a normalization of these coagulation parameters. elevation of plasma homocysteine is a hereditary disorder of methionine metabolism associated with a high risk of arterial vascular disease. however, as yet relatively little attention has been directed towards the association between hyperhomocysteinemia and juvenile venous thromboembolism (vte). consequently the aim of our study was to evaluate the prevalence of hyperhomocysteinemia (hyper-hcys) and juvenile vte. patients: 85 patients (29 men, median age 42 ys; 56 women, median age 35 ys) who had at least one verified episode of vte before the age of 45 ys were investigated in regard to their total plasma hcys levels. none of the patients had renal or liver dysfunction or evidence of any autoimmune or neoplastic disease. methods: plasma total homocysteine levels were determined by hplc with fluorescence detection. hyperhomocysteinemia was defined as hcys levels exceeding the upper limit of the normal range obtained in our laboratory from 80 healthy control subjects (40 males, median age 25 ys, hcys 95% ci: 2.02-5.67 pmol/l; 40 females, median age 27.5 ys, hcys 95% ci: 2.99-5.40 ,gruel/l). resuits: 13 out of 85 patients had hyper-hcys, giving a prevalence of 15.3 %. of these 13 patients, 9 were male and 4 female, indicating that the relation between elevated plasma hcys levels and vte may not be as strong in woman as in men. discussion: according to previous reports, our study shows that there is a high prevalence of hyper-hcys in patients with juvenile vte. however, the mechanisms by which hyper-hcys can provoke vte and whether hcys is an exclusive risk factor or if it contributes to other existing predispositions, possibly working as a trigger factor is unknown yet. some authors suggest hcys-iaduced effect on factor v activation or inhibition of thrombomodalin-dependent protein c activation. in addition an influence on thrombocyte aggregation has been postulated. conclusion: measurement of hcys levels may be useful in the evaluation of patients with a history of juvenile venous thromboembolism and could be clinically important as hyper-hcys is easily corrected by vitamin supplementation. detailed determination of the pathogenesis of vte in patients with hyper-hcys should be the aim of further investigations. a deficiency of one of the coagulation inhibitors antithrombin (at), protein c (pc) or protein s (ps) and resistance to activated protein c (apc resistance) are established risk factors for venous thromboembolism (vte). in the majority of patients with apc resistance, the .tug 506 gin mutation (factor v leiden) is present. whereas deficiencies of one of the coagulation inhibitors are rare in the normal population, the allele frequency of factor v leiden is 2-7% in western europe. heterozygous individuals have a 3-7fold, homozygous an 80fold increased risk for vte the typical clinical features of all abnormalities are deep vein thrombosis, pulmonary embolism, superficial vein thrombosis and thrombosis at unusual sites, like mesenteric vein thrombosis or cerebral vein thrombosis. the thrombotic risk is low during childhood, but increases considerably after the 13th year of age. a retrospective study in adult patients out of families with a symptomatic deficiency of at, pc or ps revealed that around 30% of surgical interventions and traumas of the lower extremities were complicated by vte. therefore, these patients should receive thrombosis prophylaxis al~er surgery and trauma if their age is higher than 13 years. pregnancy is associated with a very high risk for vte in individuals with at deficiency and prophylaxis should be initiated already in the first trimester. after delivery, thrombosis prophylaxis is adviced for all females known to have an abnormality. oc increase the risk, especially in at deficient and in homozygous factor v leiden females and are therefore contraindicated in these individuals. oc do also increase the risk for vte in patients heterozygous for factor v leiden and females known to have this abnormality should be discouraged from taking oc or should at least be informed on their increased risk. university hospital-', jerusalem, israel, hospital bcan.iou ~. paris, france, increased frequency of thrombocmbolie events have been observed iu patients with b-thalassemia. our findings of shortened platelet survival and enhanced urinary excretion ofthmmboxanc a: metabolitcs (blood 77:1749 (blood 77: , 1991 suggested an increased platclet activation in tbese patients. we also fouud that isolated thalassemie rbc enhance prothronlbin activation, suggesting an increased membrane exposure of procoagulant phospholipids i.e, phasphatidylserine (am j. hematol. 44:33, 1993) . we now show that annoxin v, which has a high specificity and affinity for anionic phospholipids inhibits pmthrombm activation by factor xa, by binding to thalassemic rbc (ic~, = 0.32 nm). kerckhoff-klinik, bad nauheim ~, medizinische poliklinik bonn 2, institut for immunologie und transfusionsmedizin universit~ll greifswald a antibody-mediated intravascular platelet activation is believed to be the basis for both arterial and venous thrombosis in patients with hat. while the development of arterial thrombosis can explained sufficiently by intravascular platelet activation, it is a matter of discussion whether additional risk factors are involved in the pathogenesis of hat-related venous thrombosis. since resistance to activated protein c (apc) is the most common inherited risk factor for venous thrombosis described the frequency of apc resistance among a population of 68 hat patients has been studied. hat was diagnosed using the heparin-induced platelet aggregation assay and confirmed by the ~4c-serotoninrelease test. the diagnosis of apc resistance was established by two functional assays and genetic analysis. at time of diagnosis of hat, 38 patients showed venous thromboembolic complications. among these, 24 were found positive for apc-resistance. pulmonary embolism was diagnosed in 18 hat patients, 14 of them were apc resistance positive. none of the 18 hat patients who showed exclusively thrombocytopenia were apc resistance positive. early oral anticoagulation (oa) was initiated in 7 patients after the diagnosis of hat has been established. six of these patients developed serious thrombotic complications including skin necrosis. these results demonstrate that apc resistance is an additional and common risk factor for the development of hat-related venous thrombosis. early initiation of oa during an acute episode of hat dramatically increases the risk of thrombosis. therefore, oa in hat patients should be initiated only after platelet counts have been returned to baseline levels and effective parenteral anticoagulation is achieved. controlled trials for primary and secondary prevention of stroke g. de (3aetano, c. cerletti and v. bertel~ consorzio mario negri sud, santa maria imbaro, italy this presentation will review the antithrombotic treatments to prevent ischemic stroke that have been evaluated in controlled clinical trials. in two studies of aspirin therapy for pdmary prevention in male physicians there was no reduction in the incidence of stroke, while that of first myocardial infarction was significantly lowered. similar results were obtained in a prospective study in a large cohort of women taking aspirin daily. the incidence of vascular death was not modified by aspirin in any of these trials. this is possibly due .to an excess of strokes associated to aspirin treatment: indeed the four vascular events avoided in 1000 us physicians under aspirin prevention for five years would result from five myocardial infarction and one vascular death avoided and two additional strokes occurred. oral anticoagulant therapy decreases the relative risk of stroke in patients with non valvular atrial fibrillation. warfarin appears to be superior to aspirin, but the latter drug is a useful alternative when long-term anticoagulant therapy cannot be administered. a metanalysis of about 150 trials and over 100,000 patients with different vascular diseases treated with aspirin (at different doses) and/or other platelet inhibitors showed 25% overall reduction of vascular events including stroke. the optimal dose of aspirin for secondary stroke prevention could not be established. in patients with previous minor strokes or tia there was 22% reduction of vascular events and 23% of non fatal strokes. the avoidance of nine strokes of any cause among the 38 expected in 1000 patients at risk would result from the sum of 10 ischemic events avoided and a haemorrhagic one occurred in excess. ticlopidine was reported to reduce the risk of stroke in two large tdals (one in patients with major stroke), but there is no evidence that it is better or safer than aspirin. we compared the effect of the direct specific thrombin inhibitors, napsagatran (na) and rec. hirudin (rh) with unfractionated heparin (uh) on the further growth of preformed thrombi. as a model of thrombogenesls, an annular perfusion chamber exposing rabbit aortic subendothelium was perfused with native rabbit blood at an arterial wall shear rate (650/s). fibrin and platelet thrombi were allowed to form during a 10min perfusion period after which the test agents were given iv as a bolus and a continuous infusion (3 and 10pg/kg/min, n=6) and the perfusion continued for 20min. the control groups were perfused for 10 or 30 rain (n=6). fibrin deposited and platelet thrombi formed on subendothelium were evaluated by microscopic morphometry. the % surface coverage with fibrin was not reduced in the drug-treated groups since fibrin deposition was similar in the 10 and 30 min control groups (39+8% and 335:6%, respectively, mean:l:sem). platelet thrombus area (ta) in the control groups increased from 24+7pm2/pm after 10min to 97+32pm2/lim after 30rain perfusion. na at 1011g/kg/min reduced ta by 94% to values (6+_2ptm2/ ~m) lower than those of the 10min control group whereas rh at this dose reduced ta by 70% (30-j:141.tm2/i.tm). uh at both doses was ineffective. these findings show that in contrast to uh the direct thrombin inhibitors na and rh inhibit the growth of preexisting thrombi. these results could be explained by the higher potency of na and rh as compared to uh for inhibiting clotbound thrombin (gast et al., blood coagul fibrinol 1994, 5.' 879-87) and suggest that thrombus-bound thrombin is an important modulator of platelet thrombus growth and/or stability in this thrombosis model. platelet adhesion -the initial event of thrombosis -is believed to be completely prevented by intact endothelium. we challenged this theory by superfusing intact human umbilical vein endothelial monolayers with activated human platelet rich plasma utilizing the stagnation point flow adhesio-aggregometer (spaa). the spaa provides flow mediated contact of platelets with the superfused surface. heparinized (3.5 -4.0 u/ml) platelet rich plasma (prp) was obtained from healthy volunteers and activated by addition of adenosine diphosphate (adp 2"10 -6 m). platelet deposition was recorded on-line by video as well as by measuring scattered light. fixed samples were examined by phase contrast and electron microscopy, inhibition experiments were performed with either the tetrapeptide rgds, the non-peptide gpiib/llla-inhibitor ro-43-8857 or a monoclonal antibody directed against the gpilb/llla complex. stimulation with adp prompted platelets to adhere to intact endothelium single or as microaggregates of a diameter of up to 100 micrometer. adhesion was dependent upon convective transport resulting in platelet collision with the endothelial monolayer. infusion of rgds or ro-43-8857 into the flowing, adp-stimulated prp completely prevented platelet adhesion to the endothelium as well as subsequent aggregation. when the inhibitor inflow was stopped while adp stimulation persisted, adhesion and aggregation occurred immediately. re-establishing the inflow of the inhibitors -with still continued adp stimulation -led to disintegration of the adhering aggregates. when prp preincubated with the monoclonal antibody against gpllb/llla was superfused, platelet adhesion to the endothelium and aggregation were irreversibly blocked. our results suggest that convective transport and stimulation of platelets are prerequisites to overcome endothelial thromboresistance and that subsequent platelet adhesion to the endothelium is mediated via the platelet gpilb/llla receptor complex. prevent thrombus formation affer ptca i.p. 8tepanova t, g.v. bashkov 2, l.p.kapralova, 2 s.p. domogatsky ~ cardiology research center t and national haematology scientific cettter 2 russian academy of medical sciences, moscow, russia percutaneous transluminal coronary angioplasty (ptca) results in atheroselerotie plague rapture, vascular wall damage and thrombogenic collagen exposure. subendothelial collagen type i-lll is a very ~rong agonist of platelet-dependent thrombus formation in arteries. the anlithrombotic action of rabbit polyclonal antibodies to rat collagen type i-ill and their chemically synthetized conjugate with monoclonals to human recombinant two-chain/one-chain urokinase type plasminogen activator (rtcu-pa/rseu-pa), cross reacting with rat tcu-pa/scu-pa was studied both an in vifro and in vivo. anticollagen antibodies and bispecific conjugate inhibited human platelet adhesion, aggregation and formation of thrombi-like ~ructures induced by rat collagen immobilized with the polystiroi surface in a condition mimics the high shear rate in the large elastic-type arteries. the short-term treatment of the collagen-soaked silk thread by the collagen antibodies suppressed the platelet-dependent thrombus formation in the arterio-venous shunt in rats by 56_+4 % (p<0.001) as well as by the bispecific conjugate (44_+4%, p<0.001). the treatment of collagen-adsorbed conjugate by rtcu-pa did not increase the autithrombotic effect of bifunctional antibodies. the present date suggest, that the local administration of the anticollagen antibodies at the site of atherosclerotic plague rapture may tm the efficient tool for prophylaxis of platelet-dependent thrombus formation in arteries after ptca. increased levels of certain hemostatic factors have been shown to be related to an increased risk of cardiovascular events. hypercoagulability is suggested to predispose to arterial thrombosis and thereby to participate in atherogcncsis. we therefore assessed fibrinogen, prothrombin fragment 1+2 (fi+2) and yon willebrand factor (vwf) antigen in 80 consecutive patients (aged 59+5 years) with known coronary artery disease (cad) who all underwent coronary angiography. the extent of coronary artery disease was quantified according to modified criteria of the american heart association (total, proximal and distal "score"). furthermore the intima-media thickness (imt) was determined in the carotid and femoral arteries by standardized ultrasonographie measurement, vwf antigen was found to correlate positively with the total and proximal coronary score (r=029, p<0.05 and r=o.36, p< 0.01). while fi+2 showed no correlation with the coronary scores, it was significantly correlated with the imt values in the carotid arteries (r=0.27. p< 0.05). after differentiating tertiles of the parameters patients belonging to the upper tertile of fi+2 concentrations had significantly higher imt values of the carotid and femoral arteries (0.81_+0.11 mm vs. 0.72+_0.13 mm in the lower tertile, p<0.05:1.38_+0.44 mm vs. i. 15-+0.25 ram, p=0.05) whereas in patients belonging to the upper tertile of vwf antigen concentrations the proximal coronary artery score was significantly higher (!.71-+0.59 vs. 1.31+ 0.92 in the lower fertile, p<0.01). fro correlation of fibrinogen concentrations and extent of cad or imt values of the carotid and femoral arteries could be demonstrated. in conclusion procoagnlatory mechanisms as indicated by elevated concentrations of yon willehrand factor antigen and fi+2 may be contributing factors in atherogenesis. we have previously shown that pgei is a potent inhibitor of pdgf-ioducod proliferation of vascniar smooth muscle cells (vscm) and inhibits dna replication by a camp-related mechanism (grol~er et al, 1994) . the present study investigates of whether or not this aatimitogeni¢ activity of pget can be amplified by trapidil, a compound that has been shown recently to inhibit the incidence of restenosis of hmnan coronary arteries subsequenmt to ptca (maresta et al. 1994) , vsmc were prepared from coronary arteries of adult bovine hearts, passagod and kept under standard tissue culture conditions. cells of passage 4-9 wore incubated in serumfree medium for 24h in the presence of indomethacin (3 p.m). addition of pdgf-bb (10 ng/ml) under these conditions stimulated dna-replication as assessed from 'hthymidin lncm'poration, by 3.-4laid above control level. trapidil at 10 idvl caused a minor reduction of pdgf-induced mitogenesis whereas 10t) of the compound resulted in a marked reduction of dna replication by 69% (p < 0.05, n = 4). pgei at 0.3 nm diminished the incorporation rate by t 1% while the simultaneous administration of both pged and trapidil (100 idyll caused a significantly stronger response as seen from n reduction of ~h-thymidine incorporation rate by 82% (p < 0.05, n = 4). as a possible mechanism of action, trapidil might have inhibited phosphodiesterases. to establish this, we measured the camp-depcudont proteinkiaasc (pk) a activity in cell homogenates. trapfdil increased the basal fka-activity from 19% to 31% of the maximum response while the response to pget (10 am) amounted to 55%. coincubation of pgei with trapidil caused a 65% stimulation of pka activity, sugesting a small though detectable inhibition of vscm phosphodiesterases by trapidil at anttmitogenic concentrations. essentially similar results wore obtained when thrombin was used as the mitogenic agent. the data demonstrate a significant antimitogenic effect of trapidil at p.molar concentrations that are in the range of plasma levels after therapeutic administration of the compound in rive. at these concentratrations, pget induced inhibition of mitogenesis is markedly enhanced by trapidil. inc. i~enna, and ~cenlral itematnlogy laboroto~. , university hospital of bern pibrinogen (fg), yon willebrand factor antigen (vwf) and tissue-type plasminogeu activator antigen (l-pal have recently been shown in be independent risk factors for subsequent coronary events in patients with angina pectoris (nejm 1995; 332:635) although paul antigen has also been proposed as a risk factor, conclusive dam showing its predictive value is still lacking. furthermore, we have recently shown in a study investigating 200 survivors of myocardial infarction that not only are fg, t-pa and pai-i significantly increased in these patients when compared to a heahhy conlro[ group, but pci activity is also elevated (7hrornb. tfaemost. 1995;73:1137 abst.) , hi order to obtain cut.off points for the individual parameters, frequency histogram plotl; were transformed into straight line cumulative frequency (probit) plots (thromb i/aemost. 1995;74:718) . the cut-off valu~ for the four parameters were determined as follows: fg at 2.7 g/l, t-pa at 8.7 ng/ml, pal-i at 25 ng/ml and pc[ at 126% of a normal pooled plasma. utilising there cut.off points it was then possible to determine the accumulative discriminatow effectiveness of the parameters. when fg w;qs employed alone as the discriminatow factor, it was observed that 47% (941200) of the coronary heart dir, ease (chd) group eilher had the cul..off value or were below it aud 29% (29199) of the normal group were above the cut-off value, thus, resulting in 47% false ne$atives and 29% false positives. when a second additional risk factor, t-pa wa_~ introduced, the number of false negatives dropped to 21% [i.e. 79% (158/200) had two, risk factors elevated] and the number of false positives to 13% to investigate whether a third parameter could discriminate further, pai-i antigen was used to analyse the rcnudning false positives and negatives. an additional 4% could be detected, resuhing in 83% of the chd group having three risk factors elevated. similarly, the number of normal aubjecta with three parameters elevated dropped by 4% to 9% furthermore. when a fourth parameter was introduced, namely pci, it was round to discriminate a further 8% in the chd group, thereby increasing tile di~riminalion to 91%. the number of false positives dropped to 4%, additionally, determination of pci increased the discrimination of patienta having had multiple infarctions from 88°/= when thrce parameters were mcasured to 100%. from these results it can be concloded that determination of fibrinogen levels alone is not sumcicnt to separate patients from controls as t-pa adds significant discrimination. pai-i antigen which correlated strongly with t-pa did not significantly increase the discriminatory potential of both fg and i-pa. however, by employing pci as a fourth paramctcr, virtually complete separation between the chd and normal groups as well as rurthcr recoguitiou of' patients having had multiple infarctions could be obtained. to test the hypothesis that oral contraceptives (oc) enhance exercise-induced activation of blood coagulation we examined 11 women (27 + 3 (sd) years, bmi 20.4 + 2.0 kg/m 2, vozm.. 49 + 7 ml/kg/min) without oc between day 18 and 22 of the menstrual cycle and 10 women (24 + 2 (sd) years, bmi 20,6 ± 1,6 kg/m', vo2max 47 + 7 ml/kg/min) taking oc (150 mg desogestrel and 30 mg ethinylestradiol) between day 7 and 21 of drug intake. prothrombin fragment 1 +2 (ptf1 + 2) and fibrtnopeptide a (fpa) were measured before and after running for one hour on a treadmill at a speed corresponding to the anaerobic threshold. mean heart rate [191 ± 10 vs. 196 ± 12 min 1) and mean plasma lactate (3.3 ± 1.6 vs. 3.1 + 1.2 mmot/i) wera comparable during exercise between control and oc group, respectively. results for markers of thrombin and fibrin formation were: ptf1 +2 (nmol/i) fpa (ng/ml) control before 0,66 ± 0.19 1.0 + 0.2 after 0.73 + 0.23 2.2 + 1.2" oc before 0.82 + 0.31 1.0 + 0.2 after 1.11 + 0.48* + 5.8 -+ 6.0* + * p < 0.05 vs. baseline, + p < 0.05 between groups. we conclude that oral contraception with 150 mg desogestrel and 30 mg ethinylestradiol enhances exercise-induced thrombin and fibrin formation, our data suggest that exercise testing might be useful for evaluating the risk of thrombosis associated with different compositions of oc. a. haushofer +, wm. halbmayer +, j. radek +, m. dittel *, r. spiel *, h prachar *, j. mtczoch *, m. fischer + + zentraltaboratorium mit thrombose-und c~rinnungsambulanz -krankenhaus lai~: * 4. medizinische ab[eilung mtt ka~liolo$i¢ -krankenhaus lainz und ludwig bottzmann-lnstitut ftlr herzinfarktforsohung, wien fifty-one patients (age 61.6 ± 9.2a; 34 m / 171) implanted with 74 coronary stems 03 palm~-schatz, 27 gianturco-roubin, 14 micro stcnts) received a now antithrombotic treatment using a combination of ti¢lopidine (tic) 2 ×250 mg/d for 28 days and acetyl salicylic acid (asa) zoo mg/d for long-term treatment. patients (pat) only received 15000 tu standard hepartn as i.v. bolus immediately before stent implantation (day l ). side effects and changes in hematological (day i to 7, 14. 28 and 35 [= without t[cil liver and kidney parameters (day 7, 14, 28, 35) were monitored. thirty-eight pat (75%) came for the controls to our del~rtment and were additionally monitored by thromboelastograpy (teg) and bleeding time (bt) (day 2g and 35). the other pat were monitored externally, side effects were reported. thrombin geucration after stenting was monitored from day i to 7 by prothrombin fragment 1+2 (f 1+2) and thrombin-antithrombin-lll-comptex (tat). "k" of the "leg decreased (day 28 vs 35; p< 0.0l ). bt prolongation was negatively correlated with the bodysurf ace area (tic+asa: p< 0.05, asa: p< 0.0 l) and showed a reduction after withdrawal of tic (2 l0 sec, 180/ 300 so: [median, quartiles] vs. 120 sec, 881157 sec; p< 0.ix)01). f 1+2 and tat of day i (blood collection: 0, 2, 4, 6 h after intervention, f 1+2:0.84 nmol/i, 0.68/i.07 nmol/l: tat: 2.6 pg/1, 2.0/4.6 ilg/1) were lower compared to day 2 to 7 (f i +2: 1.31 nmol/l, ].08/i .62 nmol/l; tat: 4.8 pg/i, 3.2/7.2 ijg/1; p< 0.0001). tic scorns not to be a strong thrombin generation inhibitor. during stenting one pat (i.9%) sustained a non penetrating mci and one (1.9%) an ischaemic stroke. tic+asa were very effective, only with one pat (1.9%) stent thrombosis (acute) occurred. side effects: 8/15.7% gastrointestinal (one lead to hospitalization), 5/9.8% hematomas at the needle site in the groin (one surgical intervention), 5/9.8% leucopcnias (one agranulozytosis with hospitalization), 3/5.9% allergic skin reactions and 2/3.9% increased liver enzymes (got, gpt, "pgt, alkaline phosphatase; > 2 × of the j. ). with one pat with gastrointestinal disturbances and skin reactions tic had to be withdrawn and treatment was changed to oral anticoagulatlon + asa. one pat showed a combination of skin reactions, gastrointestinal distufl~aneas and on day 28 a heavy reaction of the liver enzymes ( j. after 5 weeks). a decrease of the white blood count (day 1:7.19 gh, 6.03/8.2 g/l, day 28:5.46 g/l, 4.63/6.47 g/l; p< 0.000 i) could be observed. the safety of the therapy with tic+asa should be elucidated and extensively discussed. the serpins c1 esterase inhibitor (cllnh), antithrombin iii (atiii), alantitrypsin (slat), and a2-antiplasmin (azap) are known inhibitors of coagulation factor xla (fxla). although initial studies suggested al at to be the main inhibitor of fxla, we recently demonstrated cllnh to be a predominant inhibitor of fxla in vitro in human plasma (wuillemin et el., blood 1995; 85:1517) . the present study was performed to investigate the plasma elimination kinetics of human fxla-fxla inhibitor complexes injected in rats. the amounts of complexes remaining in circulation were measured using elisas. the plasma tl/2 of clearance was 98 min for fxla-alat complexes, whereas it was 19, 1 8, and 1 5 min for fxla-cllnh, fxla-a2ap, and fxla-atiii complexes, respectively. thus, due to this different plasma tl/2, preferentially fxla-alat complexes may be detected in clinical samples. this was indeed shown in plasma samples from thirteen children with meningococcal septic shock (mss), a clinical syndrome which is complicated by activation of coagulation, fibrinolytic, and complement systems. fxla-fxla inhibitor complexes were assessed upon admittance to the intensive care unit. fxla-a 1at complexes were elevated in all patients, fxla-c11nh complexes in nine, fxla-atiii complexes in one patient, and no elevated fxla-a2ap complexes were found. we conclude from this study that, (1) although c1 inh is the predominant fxla inhibitor, fxla-alat complexes may be the best parameter to assess activation of fxi in clinical samples, (2) measuring fxla-fxla inhibitor complexes in patient samples may not help to clarify the relative contribution of the individual serpins to inactivation of fxla in rive, and (3) fxl is activated in patients with meningococcal septic shock. dudng the coagulation of plasma about 20 % of the (x2ap present is covalently crosslinked to fibrin by factor xiila (aoki und sakata 1980, thomb. res. 19: 149-155) . we investigated the binding of azap by factor xiila to soluble fibrin (desaabb-fibdno) whose polymerization was inhibited by an isolated fibrin ddomain named d=,,, (haverkate and tiemann 1977, thromb. res. 10: 803-812) . d==. is known to have an intact fibrin-polymerization site and is able to block the prolongation of the fibrin protofibrils at an early stage depending on its concentration. lateral association to fibrin fibers does not take place, since the inhibited protofibnls formed at the conditions used here do not reach a sufficient length (williams et el. 1981, biochem. j. 197: 661-668; hantgan et al. 1983, ann. n. y. acad sci. 408: 344-365) . material and method: soluble desaabb-fibrino was prepared by incubation of (lztl)-fibrinogen (0.48 mg/ml), d=1o (1.91 mg/ml; molar ratio d==o to fibrin 14:1) and 0.4 u/ml thmmbin for 45 min. then q2sl)-c~2ap (16 p.g/ml), faktor ×ill (2 ulml) and ca 2) (5 mmol/i) were added. the crosslinking reaction was stopped at different times of factor xiila-incubation by adding of urea/edtasolution. the suspension was analysed by ultracentrifugation on gradients containing saccharose, urea and sos. re~ultl: the elution profiles of the ultrecentifugation-gradients show the formation of cmsslinked fibrin oligomers of increasing size depending on the time of factor xiila-action. the crosslinked fibrin polymers contained about 80% of the fibrin initially added. although factor xiila acted well, crosslinking of azap in the fibrin oligomers could not be observed. conclusl0n: as we already demonstrated (kelach et el. 1994, ann, hematol. 70 (suppll) : a 60) the crosslinking of azap to fibnn clots depends on the structure of the fibdn network, especially on the degree of lateral association of the fibrin pmtofibdla. in desaabb-fibrino no lateral association of fibrin protofibnls takes place under the conditions chosen here. thus it is consistent with our theory that we did not observe any binding of aiap to the fibrin oligomers of desaabb-fibrino. human pci is a non-specific serpin that inhibits several proteases of the coagulation and fibrinolytic systems as well as tissue kallikrein and the sperm protease acrosin. it is synthesized in many organs including liver, pancreas, and testis. the physiological role of pci has not been defined yet. recently, we have cloned and sequenced the mouse pci gene (zechmeister-machhart etal., manuscript in prep.) . this enabled us to study pci gone expression in murino tissues using mouse pci edna and crna probes. by northern blot analysis, mouse pci tar.ha was exclusively found in the reproductive tract (testis, seminal vesicle, ovary), all other organs analyzed -including the liver were negative for pci mkna, indicating that in the mouse pci is not a plasma protein. to determine which cells of the reproductive tract synthesize pci, cellular localization was assessed by in situ hybridization of mouse testis and ovary sections. in testis, pci mrna was present in the spermstogonia layer and in leydig cells, while sertoli cells and peritubular myoid cells were negative. these results are consistent with the immunohistological localization of human pc1 (laurell et al,, 1992) . in the mouse ovary, stroma cells of the medulla and around the follicles were positive for pci mrna. no pci expression was detected in theca or granulosa cells. we also studied the regulation of mouse pci gone expression by steroid hormones in vivo. [n mature male mice castration caused an increase in pci mrna in seminal vesicles, which was reversible upon the administration of testosterone. in tissues of intact adult male and female mice, pci mrna levels decreased after injection of human chorionic gonedotropin (hcg), while in castrated male mice, hco had no effect on seminal vesicle pci mrna. progesterone and 17-b estradiol decreased ovarian pci mrna levels in immature female mice. these data suggest direct down-regulation of mouse pci by sex steroids. the different tissue specific pci-geoe expression in men and mice furthermore indicates a different biological role of this serpin in the two species. ctr. transgene technology, leuven "[' 1-tissue factor ('it) is a 47 kda glycoprotein mainly known a the primary cellular initiator of blood coagulation. whether tf expression may also play a role in development is unknown, but the lack of spontaneous viable mutations of the tf gene in rive leads to the speculation that its absence may not be compatible with normal embryonic development. to determine the significance of "if in ontogenesis, the pattern of tf expression in mouse development was examined and compared to the 'if distribution in human postlmplantation embryos and fetuses of corresponding gestational age. at early embryonic period of both murine (6.5 and 7.5 pc) and human (stage 5) development there is a strong tf expression in both ectodermal and entodermal cells. "if decoration was seen during ontogenetic development in tissues such as epidermis, myocardium, bronchial epithelium, and hepatocytes, which express "if in the adult organism. surprisingly, during renal development and in adult organism tf expression differs between men and mice. in humans maturing stage glomerali were "if positive whereas in mice glomeruli were negative and instead epithelia of tubular segments were tf positive. in ncuroepithelial cells there was a striking 'if expression indicating a possible role of'if in neumlation. moreover, there was a robust tf expression in tissues such as skeletal muscle, and pancreas, which do not express in adult. in contrast to tf, its physiologic ligand factor vii was not expressed in early stages of human embryogenesis, but was detectable in fetal liver, the temporal and spatial pattern of tf expression during murine and human development support the hypothesis, that 'if serves as an important mo~hojzenic factor darinz embrvozenesis. to serve as an anticoagulant, protein c (pc) must be activated by a complex formed between the enzyme thrombin (t) and its cofactor thrombomodulin (tm). therefore, downregulation of endothelial cell surface expressed tm, for example, triggered by an inflammatory stimulus, could become a critical factor in effective pc activation. in order to develop a recombinant (r) pc mutant which can be activated independently of the tkm-complex, a peptide sequence including p1-7 in the activation peptide of pc was modified to be identical to the factor xa (fxa)-cleavage site in prothrombin. the mutant was expressed in hu 293 cells, purified and its anticoagulant properties characterized. using purified fxa the mutant showed activation rates between 0.02 and 0.13 nmlmin at pc concentrations between 97 and 770 nm, while the rpc wild type was insensitive for fxa activation. the activation reaction is calcium-dependent reaching maximal activation rates at a calcium concentration of 3 mm and was enhanced to 3.8-fold by addition of anionic phospholipids (pl). in contrast to the wild type pc the rpc mutant was insensitive for activation by the t/i-m complex. addition of the mutant to normal human plasma induces a prolongation of tissue-factor and p-it-based clotting assays. using normal human plasma as a source for fxa the the activation rates of the mutant were found 5-fold higher than in the purified system if tissue factor was used to generate fxa. in conclusion, our data demonstrate that the rpc mutant is effectively activated by fxa in a purified as well as in a plasma system. interestingly, the activation rates are enhanced in the presence of pl and normal human plasma. fudher studies should clarify the potential use of this mutant as a novel anticoagulant. thrombln plays a pivotal role in thrombotic events. the time course of thrombln concentration in blood or plasma after activation is of special interest to answer a variety of questions. with a chromogenic assay developed by hemker et el. [thromb. haemostas, 70, 617, 1993] it became possible to measure the generation of thrombin in activated plasma continuously. inhibitors of clotting enzymes which are to be developed as anticoagulants should be able to inhibit thrombin generation or to immediately block generated thrombin. we have used a test based on hemker's thrombin generation assay to elucidate which potency and specificity an inhibitor of factor xa needs to efficiently block thrombin generation in human plasma. thrombin generation after extrinsic (tissue factor) or intrinsic (ellagic acid) activation was followed using the chromogenic substrate h-~ala-gly-arg-pna (pentapharm ltd.). a series of synthetic low molecular weight inhibitors as well as naturally occurring inhibitors of factor xa with different potency were investigated. because of the inhibition of activated factor x the generation of thrombin in plasma is delayed and the amount of the generated thrombin is reduced. the concentrations which cause a 50 % inhibition of thrombin generation (icso) correlate with the k~ values of the inhibitors. low molecular weight inhibitors with k~ values of about 20 nmol/i inhibit the generation of thrombin after extrinsic activation with icso in micromolar range. after activation of the intrinsic pathway tenfold lower concentrations are effective. the strongest inhibitory activity after extrinsic as well as intrinsic activation is shown by recombinant tick anticoagulant peptide (r-tap) with ic~o of 0.049 pmol/i (axtdqsic) and 0.037 pmo/i (intdnsic). in the compadson of synthetic low molecular weight inhibitors of thrombin end factor xa which have similar k= values for the inhibition of the respective enzyme (lowest i<1 20 nmol/i), factor xa inhibitors are less effective tn the thrombin generation assay. in contrast, the highly potent xa inhibitor r-tap shows a stronger inhibition of thrombin generation than the tight binding thrombin inhibitor hirudin. background: resistance to degradation of coagulation factor v by activated protein c is associated with a point mutation in which adenine is substituted for guanine at nucleotide 1691 in the gene coding for factor v. to date this specifc mutation appears to be the most common inherited abnormality which predisposes patients to venous thrombosis. for this reason a reliable, fast and automatable system for the diagnosis of the described point mutation is required. the conventional methods used to identify the mutation are based on allele-specific restriction enzyme site analysis or direct sequencing. these methods have disadvantages for a large scale dna diagnosis, which include the need for electrophoresis or a high cost and time consumption. methods: an alternative strategy of dna diagnosis, the allele-specific oligonucleotide ligation assay, was adapted for the diagnosis of tile point mutation of factor v. following pcr amplification of the target dna, tile procedure was performed completely automatically on a robotic workstation with an integrated elisa reader using a 96-well microtiter plate. allelespecific restriction enzyme site analysis was performed to confirm the genotypes. results: in 10 patients with the mutation and in 20 individuals without the mutation the genotypes determined with the conventional allele-specific restriction enzyme site analysis were in 100% concordance with the elisabased oligonucleotide ligation assay. discussion: the pck-oligonucleotide ligation assay applied as automated detection system for the identification of the coagul;mon factor v point mutation allows tile rapid, reliable, and large scale analysis of patients at risk for thrombosis. resistance to the asticoa=m~lant activity of activated protein c (apc resistance) has emerged as the most con'anon inherited thrombophilic state. patients lreterozygous for factor v leiden are more likely to suffer from thromboembolie events than controls. this risk is even more pronounced in homozygotes. due to the low sensitivity and speeifity of most coagulation tests some investigators suggest to examine patients for the presence of factor v leiden mutation by pcr-based methods. re e~tly we presented an aptt-based functional test (acceleria inactivation test ait): 1:20 diluted plasma (50bi) is mixed with factor v deficient plasma (50~tl) and aptt reagent (501.d), incubated at 37°c and then coagulation is induced by caci2 and a.pc (50~d). using a standard curve, the clotting time (see) is transferred in per cent accelerin inactivation (%ai). using this test, the widely used apc-ratio as well as pcr-based factor v leiden detection (confirmed by direct sequencing) we prospectively studied 172 consecutive patients with thromboembolic events. patients without the factor v mntation eonsitently showed more flazm 50% al with the exception of one patient with severe factor deficiencies (including factor v) due to hepatic failure and heterozygous for factor v-leiden resulting in 62*/. ai, there was a complete concordance between the pcrbased method and dysaseelerinemia detected by ait. due to these result a specifity and sensitivity of ait above 95 % was calculated. furthermore, a clear discrimination could be obsoved beween 34 heterozygotes (20%<ai<50%) and 8 (incl. 5 samples obtained from jeaa) homozygotes (ai<20%). in contrast a.pc-ratio resulted in a great overlap between patients homozygous for the wildtype , hcterozgous or homozygous for factor v leiden. in addition we confirmed thc di.~eulties nsing the apc-rauo for patients trader anticoagulation, or with different types of other d~rombophilie defects such as lupus anticoagulant or protein s deficiency. due to the time and cost-saving featttres the/kit should be considered for the rapid detection of apc resistance without the use of pcr-based techniques. in summary we found, well in accordance with previous reports, that apcr due to the fv-leiden mutation is • highly prevalent defect in vte pts. pts with the fv-leiden mutation were eharacterised by a higher incidence of vte's without recognised prothrombotic risk factors and more frequent positive family history; no higher incidence of recurrent vte's or other prothrombotie coagulation defects was found; the age at initial manifestation of thrombophitia was comparable. acquired a recently identified mechanism for famdial thrombophilia is characterized by a poor anticoagulant response to actavated protein c (apc), it is now welt recoginzed to be due to a genetic defect in the factor v gene, which results in the syn-,thesis of an abnormal factor v molecule and to be a frequent risk factor for thromb.osis, however apc resistenee can also be found in certain acquired conditions. since pregnancy ~s known to be associated with a hypercongulable state resulting in a prevalance of thrombosis up to 1% and thromboembolic events, we determined the prevalence of apc resistance during the course of pregnancy at the city hoslfital ruesselsbeim. germany. blood samples from pregnant woman were drawn at mmester 1, 2, 3 and at term. besides the routine coagulatton screening, protein c and viii:c, the apc resistance were evaluated. a modified aptt method utilizing human plasma fracnonated apc (amencan red cross. bethesda, maryland, ~usa) was used to assay apc resistance. this method relies on the determination of two aptt's in the patient plasma, one in the absence of apc and one in the presence of a fixed amount of apc. clotting tune was determined after mcubation using the aptt reagent ( we investigated the sensitivity of various species towards activated, human protein c (apc) and found an impaired response in the order monkey < horse < pig < dog < rabbit. we assume that this effect is mainly caused by differences in the apc cleavage region of factor v, as recently described for a factor v mutation in bumat~s: factor v-leiden. this was corroborated by the finding that addition of human factor v similar to the factor v added with the animal plasma had only minor influence on test outcome. coagulation assays dependent on apc, such as for protein c and protein s determination, in the standard apc assay, the more factor v-leiden specific apc assay and in the pcat, a new screening assay for the whole protein c system, are based on the inactivation of factor va via apc. thus, in all these assays, 5% of rabbit plasma added to human plasma mimicked a apc response as observed for heterozygous carriers of factor v-leiden and 20% as for homozygous carriers. we exploited this factor v-leiden like behaviour to prepare plasma standards for calibration of apc dependent assays by mixing rabbit plasma with human plasma~ as reference for these standards the 0% value was assigned to the clotting time in the absence of apc, while the 100% value was assigned to the clotting time in presence of apc as obtained with a human, normal plasma pool. then these calibrated standards were used for establishing reference curves. on automated coagulation analyzers, the results obtained were automatically reported in '% of normal' or '% sensitivity'• this not only eases evaluation, more importantly, this procedure also considerably improves the comparability of results obtained with different reagents and instruments. in order to establish the methods for activated protein c resistance and the faktor v leiden-mutation we investigated 204 healthy individuals (128 women, 76 men, median age 37 ys) using the assay "contest ® apc-resistance" (chromogenix, sweden). the mean apc-ratio was 2.65, the cut-off level for low response to apc was calculated as 2.15, 46/128 women were oral-contraceptive users. they showed a slight decreased apc-ratio (mean 2.46). 24 of the 204 individuals (11,8%) had a poor response to apc (mean ratio 1.94) as a apc-resistance. for detecting the factor v gene mutation we a used non commercial own modified easy to handle pcr-based test. in 14 of the 204 individuals (6.9%) the factor v-leiden mutation could be found as heterozygotes. in this group, the apc-ratio was significant lower (mean 1.87) as in the group with apc-resistanee and without deteetiun of the factor v mutation (mean 2.04). nevertheless in one individual with beterozygote factor v-leiden, the apc-ratio was found in the normal range (2.24) , also in a few individuals with very iow apc-ratio the factor v mutation could not he detected. finally this data suggests a high prevalence of apc-resistance and factor v-leiden mutation in the population of the southwest of germany. resistance to activated protein c (apc) has been reported to interfere in clotting assays for protein c (pc) and protein s (ps) from various manufacturers, resulting in decreased values or even apparent deficiency (type ii') of pc. or is. in our laboratory, when using functional, avit-based assay kits for pc., is, and apc sensitivity from behring (marburg, germany) on an automatic turbidimctric coagulation analyzer (fibrintiraer a, behring), the interference in the is assay has been observed in a very pronounced manner soon after the introduction of apc sensitivity testing into the standard laboratory procedure for patients with thrumbophilia. yet we were not aware of an interferenca caused by apc resistance in the pc test. we therefore evaluated our results for pc eed ps with respnet to apc sensitivity status in a retrospective analysis. exclusion criteria were: oral anticoagulant therapy with vitamin k antagonists, impaired liver function, borderline result in the apc sensitivity test, known analytical intedercnc* other than apc resistance (e.g. elevated factor viii:c). resu/ts: apparant pc activity was slightly (12 %) lower and apparent ps activity was grossly (45 %) lower in ape resistent patients than in patients with normal apc sensitivity: there was no case with a presumed du~l defect of both at~ rnsistaac¢ and pc deficiency. in addition, none of the few patients diagnosed with pc deficiency before the introduction of the apc sensitivity assay turned out to ix: apc resistent. much more of a problem is the known sensitivity of the pc test to an elevated factor viii concentration which is quite a common finding among hospital patients but rarely known in an individual case. by aeccelerating the clotting process, elevated factor viii can cause apparent pc deficiency that can not be confirmed in a chromogunie pc assay. ,clearly, when combined with apc resistance, this problem is exspacted to be more severe. on the other hand, the comhination of both apc resistance and apparent is deficiency was found on a regular basis with many decreased and some bordeflinc values for is but only few in the lower normal range and virtually none in the upper non~al range. also, several #eats previously diagnosed with is deficiency turned out to be pc resistent, and the diagnosis had to be corrected, but dual defects cannot be ruled out. conclusion: for, the interpretation of pc and/or 1:"3 values from clotting assays the apc sensitivity status must be considered. when apc resistance and an apparent deficiency of pc or is is found, a type i deficiency may be confirmed or ruled out using antigen assays, but at present, there seems to be no reliable way other than genetic analysis to address the question of dual defects with presumed type ii deficiency, especially in the case of ps. therefore, there is a clear need for analytical improvements and the development of functional assays that are less sensitive to interference by apc resistance. the most common inherited risk factor for venous thrombosis is the resistance to activated protein c (apc). the main reason for this resistance is a point mutation in the gene of dotting factor v (f v). a nueleotide exchange leads to a different amino acid sequence in the protein and thus to the loss of one of the cleavage sides which is important for the inactivation of the procoagulatory form off v. the imbalance in the coagalation cascade leads to a strong risk for venous thrombosis, which is thought to be inoreaced by factor 7 to 10 for heterocygotes and about 80 for homocygote individuals. the prevalence in patients with a history of thrombosis is about 20%, among apcresistant patients as high as 80%. prevalence-off v ,leiden" is reported to be 2% of the normal population in leiden/netherlands and as high as 15% in a certain city in the south of sweden. due to the obviously different geographic distribution and the highly increased risk of venous thrombosis in carriers of the mutation investigated the distribution of this mutation in the normal population of different regions in germany. first attempts to assess the prevalence off v mutation by molecular methods (polymerase chain reaction followed by restriction fi'agment length polymorphism analysis) revealed that in the area of wiirzburg 7.7% of blood donors carry the mutation, which is significantly different (fischer t-test, level of significance 0.05%) from published data observed in freiburg (3.4%). homburg/saar and harmover revealed 5.8% and 8.5%, respectively, and were not significantly different from wfwzborg. further investigations of blood somples fi:om other regions should give an overview of the geographic distribution of this mutation in germany and might finally help to draw a map era regional risk of venous thrombosis. the regular apc resistance test is not applicable to plasma from orally anticoagulated (oac) or heparinized patients due to decreased levels of vitamin k-dependent clotting factors and to thrombin inhibition by antithrombin, respectively. the former limitation is overcome by prediluting samples 1-1-4 with fv-defident plasma. for this purpose a special quality of fv-deficient plasma has been prepared, which also allows analysis of heparinized plasmas. this fv-deficient plasma has now been evaluated with the coatest apc resistance assay. a complete ape ratio discrimination for the fv q506 mutation is obtained for plasmas from oac and heparin patients as well as from non-treated individuals. ratios above 2.2 are obtained in normal genotype plasmas, whereas heterozygotes for the mutation yield ratios in the range 1.6-2.0. the basal apt times for 15 oacplasmas with widely spread inr-values were reestablished into the normal range. .the stability of the reconstituted fv-deficient plasma was found to be' at least 3 hours at room tempetature, indicating its suitability for .routin.e u.~. f .u~ .e~mv~., the influence of preanalytical procedures is rmm~ single or double cenuifugation as well as freezing of samples do not alter the apc ratios to any significant extent. our results prove the usefulness of the modified coatest apc resistance kit method as a reliable, simple and rapid tool when screening for the presence of the fv q506 mutation. hereditary resistance against the anticoagulatory action of activated protein c (apc-resistance, apcr) which was first described by dahlb~ick et al. (pnas, 90:1004 , 1993 was identified as a possible new thrombophilic factor in a high percentage (17-60%) of young adults with thrombotic events. a single missense mutation (r506q) due to a g/a transition (g1691a) in exon 10 of the factor v gene (bertina et al., nature, 369:64, 1994) is tightly linked with apcr and is regarded as the causative molecular defect. identification of this mutation by pcr-based methods is easy to perform and avoids preanalytie and analytic errors in the eoagulometric assay for apcr. since the impact of this mutation in children with thromboembolic disease has not been determined to date, we initiated a multi-center prevalence study on two pediatric populations, with and without thromboembolie events. we compared 125 pediatric patients with thrombosis, divided into 3 different age groups (0 to <0,5 years; >0,5 to <10 years; >10 to <18 years) with a normal population of 159 children. the mutation g1691a was found with an unexpected high prevalenee of 12% in our normal controls. however, the prevalence was significantly higher in the age groups: 0 to< 0,5 years (25%) and >10 to <18 years (30%). in patients between >0,5 to <10 years the overall prevalence was similar to the control (13%). however in patients of this age with spontaneous thrombosis apcr was also a significant risk factor (29%). our results emphasize the impact of apcr for thrombogenesis in children. however, the significance is agedependent and does possibly reflect the different physiology of haemostasis in our three age groups. activated protein c (apc)-resistanec is a newly reeognised risk factor for thrombosis. in at least 90% of the cases it is caused by a single point mutation in the factor v gene (g->a at nucleotide 1691), which predicts replacement of arginin 506 with ghitamin. one of the apc cleavage sites in factor va is located c-terminal of arginin 506, and mutated factor va (factor v leiden) is resistant to apc-mediated inactivation. from epidemiologic studies it is known, that this abnormality can be found in about one third of patients with thrombosis. apc-resistance is a major basis for venous thromboembolism and is prevalent in about 5.10% of the general caucasian population. recurrent spontaneous abortion (rsa) affects 1-5% of couples and represents a major concern for reproductive medicine. in spite of extensive endocrine, genetic, serologic and anatomic evaluation some 30-40% of rsa women remain unexplained. a frequent morphologic finding in placentae of aborted pregnancies is an increase of fibrin deposition within the intervitlous space. because of these findings we studied the prevalence of apc-resistance in women with rsa (more than 3 miscarriages) of unknown origin. in 2 of 35 cases we found a pathologic apc-resistance, both patients had a history of recurrent thrombosis and were heterozygous for factor v leiden. the prevalance of apc-resistance is 5,7% and thus equals the prevalence in the general population. our data do not support the hypothesis that apc-resistanee is a risk factor for recurrent spontaneous abortion. h~matologisches zentrallabor der universit~t, inselspital, 3010 bern resistance to activated protein c (apc) due to the mutation 506 arg --~ (]in of factor v (factor v leiden mutation) is the most frequent hereditary thrombophilic defect known today, with a prevalence of 20 -50 % in patients with idiopathic venous thromboembolism and of about 3 -5 % in the general population. with an allele frequency of 2 % the expected number of homozygous individuals is about 4 in 10000. homozygous and heterozygons individuals differ considerably with respect to the relative risk of thrombosis (80 -fold versus 7 -fold) as well as to the age of the first thrombotic event (31 versus 44 years). deficiency of the vitamin k dependent protein s (p$), an important cofactor of apc, is another hereditary thrombophilia which is, however, much rarer than apc resistance with a prevalence of 5 to 8 % in patients with venous thromboembolism. factor v leiden mutation as well as ps deficiency are associated with impaired anticoagulatory activity of apc, which is most pronounced in case of the combination of the two defects. the combination of ps deficiency (with an assumed prevalence similar to that of pc deficiency) with heterozygous or homozygous apc resistance can be expected with a probability of 1: ~ 5000 or 1: ~ 500000, respectively. it is well known that ps levels decrease towards the low normal or even subnormal range during pregnancy. moreovar, there is increasing evidence that the sensitivity of plasma to the antieoagulatory effect of apc decreases during pregnancy resulting in an acquired apc resistance. these pregnancy associated effects art obviously much more relevant in case of preexisting ps deficiency or apc resistance and should contribute to the elevated thrombotic risk during pregnancy in a subject with either of the two defects, and even more so for a woman who suffers from both defects. we describe a young woman with a combination of homozygens apc resistance ( apc ratio 1.10, normal range: 2.02 -3.73), pronounced ps deficiency (free ps 0.ll u/i, total ps 0.30 u/i, normal range: 0.55 -1.20 u/1 and 0.65 -lag u/i, respectively) and, moreover, impaired fibrinolysis (no change of euglobulin -lysis time after 10 rain venous occlusion) who developed deep vein thrombosis after cesarean section in her first pregnancy. examination of her familiy showed heterozygous apc resistance in her asymptomatie father (apc -ratio 1.99) , combination of heterozygous apc resistance (apc -ratio 1.60) and ps deficiency (free ps 0.30 u/i, total ps 0.58 u/i) in her nsymptomatic mother and no defect in her sister. considering the fact that the mother was still thrombosis free at the age of 49 one may assume that the thrombosis risk in the proposita was mainly influenced by the homozygnsity for apc resistance. s. ehrenforth, m. adam, b. zwinge, i. scharrer university hospital, dept. of angiology, frankfurt a.m., germany introduction: apc resistance has been shown to be the most commonly inherited defect which constitutes a risk factor for venous thrombosis (vt). however, most of the present epidemiological studies concerning apc-r prevalence in thrombophilia were derived from results of tests conducted onplasmas collected under various conditions. this may influence the great differences reported on the prevalence of apc-r among these patients. for example, it has been shown that freezing of plasma specimens prior to analysis of apc-r causes a significant decrease in the assay results.the aim of our study was to evaluate the influence of eentrifugation conditions on the results obtained with the chromogenic apc-r assay. patients and methods: blood was collected from 31 patients (t9 women, 12 men; fv gent.type: 13 r/r 506, 14 r/q 506, 4 q/q 506) through veinpuncture into trisodmm ciwat (1:9). platelet-rieh and platelet-poor plasma was obtained by immediately centrifugation at 4"c for 10, 20, 30, 40, 50, 60 rain at 1000, 2000, 3000, 4000, 5000 and 6000 rpm. additional, pnp obtained from 14 healthy individuals (7 male, 7 female without hormonal trealraent) was prepared equally. apc-response was determined within one hour after centrifugation using the coatest apc resistance kit from chromogenix. results: for both, pnp and sin/gle plasma samples, we observed continuous higher af'c-ratios after increasing cenwifugation intensity. for example, an increase from 1000 to 6000 rpm resulted m an increased apcratio from 2.7 to 3.26 (20 min), from 2.88 to 3.326 (60 rain) respectively. even though less distinctive, similar results were observed concerning the duration ol eentrifugation: when the duration was increased from 10 to 60 minutes we observed a continuous increase in apc-ratio, for example from 2.74 to 3.12 when using 2000 rpm and from 3.09 to 3.36 when using 4000 rpm. the decrease of the ratio after low eentrifugation is the eonse-9nence of the shortening of affft in the presence of apc, without a signhcant influence of basal al:rl~ without apc. conclusion: our results demonstrate that centrifugation conditions are important to consider for the interpretation of apc-r results. supporting our observations, recent studies from sidelmann et al. have shown that an increase in plasma platelet concentration, low eentrifugation respectively, causes a signficant decrease in the apc-response. however, so far the mechanism responsible for the significant effect of both on apc-r assay results is unknown. although technically simple, the biochemical cemplexitiy inherent in the chromogenic apc-r assay necessitates a standardized plasma handling procedure to secure a reproducible determination of apc-il compapjson of different assays for determination of apc-resistance with the geno'fyping factor v (arg -> glu) g. siegert*, s. gehrisch*, e. runge**. r. naumann**, r. kn6fler*** *institute of clinical chemistry, **clinic of internal medicine, *** childrens hospital resistance to apc diagnosed on the basis of prolongated clotting time in the aptt assay" is now considered a major cause of thrombophilia. in the majority apc resistance is ~ted with a point mutation in factor v molecule (arg506glu), but both are not synonym. protongated baseline aptt is a limitation of the assay. following the determination is not possible in risk groups of patients (factor)ill deficiency, lupus anticoagulan0 and in patients under anticoagulant therapy. in these causes a dilution of plasma in factor v deficient plasma is recommended. the immunochrom assay is based on the inactivation of factor villa by apc. the aim of the study was to compare different functional apc response assays with the result of the dna analysis. apc response was tested in 100 healthy probands, 81 thro~ patients and 16 family members using the lmmtmochrem assay, the contest (chromogeaix) and the contest with 1+ 4 dilution of the plasma in native factor v deficient plasma (immune). the dna analysis was performed as described by bertina. one patiem was homozygoas for factor v mutstion~ a hetemzygous result was obtained in 4 members of the control group, in 28 patients and in 6 family members. in all cases with factor v mutation the ratio of the immunochrom assay was lower than the laboratory own value, independent on anticoagulant therapy. pathological ratios in this assay were also obtained in one member of a family" with high thrombotic incidence (dna arg/arg) and in 17 patients under anticoagulant therapy ( two of this patients are one cloned twins). in the contest a ai~ response was diagnosed in all cases with factor v mutation without anticoagulant therapy and in 40 % of heterozygous patients under anticoaglant therapy. results of the test using the dilution in factor v deficient plasma showed a good agreement vath the results of the dna analysis but the method is obviously only sensitive for the factor v mutation. the reason for pathogical ratios in the lrnmunechrem assay in wildtype patients is unclear. the majority of this patients is treated with anticoagulants, a comparison with the contest is not possible. interestingly in one patient under heparin and low ratio in the immunochrom assay' after reduction of hepann the ratio of the coatest was also low. it seems necessary to investigate in which distance to the thrombotic events the apc resistance should be tested. following pathological ratios in ftmctional apc assays must be discussed: high levels of factor viii and or v wiuebrand antigen (acute phase reactien), other mutations in factor v and viii. the factor v dilution assay should be replaced by the dna analysis. due to their differing compositions, the "sensitivities" of various aptf reagealts differ not only with respect to factor depletions, heparin and fibrin-fibrinogen degradation products, but also with regard to pathological inhibitors. for lupus anticoagulants this means that "lupus-sensitive" reagents can be delineated from "lupus-insensitive" reagents. with a "lupus-insensitive" ai~ reagent there is no or only slight prolongation of the aptt in the plasma under investigation, whereas with a "lupus-sensitive" reagent marked prolongation is observed. for the meaninof~l use of aptr reagents it is necessary to know the extent to which they are influenced by lupus anticoagulants. the following 14 apti' reagents were tested: • ptt-reageaz, p'rta, ptta liquid, ptt-la, pti'-lt (boehringer/stago) • pathromtin, pathromfin sl, necthroratin (behring) • platelin s, piatehn excel ls (organon tekinka) • actin-fs, actin-fsl (dade) • aptt silica lye, aptt silica liquid (instntmentation laboratory) the material for investigation consisted of 20 plasmas from patients with lupus anticoagulants. a confirmatory test (lupus anticoagulant test, immune) was positive for all of the patients. measurements were made using the sta coagulation analyser (boehringer/stago). it can be seen from the results that in some instances very different prolongations were obtained in identical plasmas by using differing aptt reagents. low susceptibility to lupus anticoagulants was shown by actirt fs (dade), ptt-reagenz (bcehrlnger) and neothromfin (behring). high susceptibility was shown by platetin excel ls (organon teknika), ptt-la and pti'-lt (boehringer/stago). lupus anticoaguhant screening with the aptt reaction is promising when two aptr reagents differing as greatly as possible in their lupus anticoagulant sensitivity are used. the resistance to the anticoagulant response of activated protein c (apc) is a major cause of venous thrombosis. apcresistance is due to a single mutation in factor v gene, which predicts replacement of arg 560 in the apc-cleavage site with gln (factor v leiden mutation). in contrast to other known genetic risk factors for thrombosis, this factor v 1691 g-a mutation has a high prevalence in the common population of western europe (average 4-5 %). we have determined the prevalence of the factor v 1691 g-a mutation in a population of 700 probands of north-eastern part of germany. the mutation was found in 7 %. (heterozygoty were found in 48 subjects 1 person was homozygous.) the results are compared with our studies of populations from argentine and poland. me analysed the factor v 1691 g-a mutation in 71 patients with thrombosis from germany and hungary. this mutation has been found in about 60 % of these patients. in contrast, the frequency of this mutation was strongly reduced in a group of 47 patients with thrombosis and pulmonary embolism of argentine (3 heterozygotes in 47 patients; 6 %). the results of these different populations will be described and discussed. past medical history: venous thromboembolic events (re) at 18, 19 and 2i years; intermittent oral anticoagulation (oac) without te's. diagnosis of autoimmune disorder with elevated antinuelear-antibody-fiters and positive lupus-anticoagulant test. no other relevant illnesses; family history uneventful. two weeks prior to the referral to us -acute febrile illness with nausea, diarrhea, abdominal pain; hospitalisation, treatment with iv antibiotics and anticoagulation with fraetionated heparin; development of extensive deep vein thrombosis (dvt) of the right leg; initiation of full-dose unfractionated heparin; decline of platelet count from 121 to a nadir of 21 g/l; referral to our department. on admission an extensive coagulation screen yielded the following results (n/normal, t/elevated, i/reduced, +/positive, -/negative): pt t, aptt t, tr n, factor ii, v, viii n, factor vii, ix, xi, xii /,, fibrinogan t, atiii n, protein c, s *, activated protein c sensitivity ratio 1.92 ($), fv-leidenmutation pcr -, fibrinolytic system n, tat t, ft÷2 t, lupus anticoagulant +, heparin induced platelet antibodies +; no diagnosis of a specific autoimmuna disorder could be made. an immunosuppressive therapy with corticosteroids and anticoagulation with recombinant hirudin were init'~at~; no p~ogr~zsion of the dvt oeeured and normalisation of the platelet count was observed. during follow-up under oac ) and low-dose corticosteroids, the patient was well, the pathologic coagulat;.on results, including lupusanticoagulant and activated protein c resistance, have returned to normal; no further te's have been observed. in summary we present a case of a complex coagulation disorder as part of an autoimmune process, resulting in a clinically manifest prothrombotie dysbalance including lupus anticoagulant, acquired resistance against activated protein c and heparin induced thrombocytopenia (type ii), entering complete remission under combined immunosuppressive and anticoagulant therapy. in the last 30 years, a vast number of simplified analytical procedures have been developed for the diagnosis of haemostatic disorders. today the detection method have evolved from the mechanical hooking method or ball coagulometry to optical systems, which additionally can utilise chromogenic substrates or immunological methods. in these systems the clotting time is derived from algorithms (e.g. threshold or maximum of the first or second integral). we studied 50 healthy subjects, aged 21 to 65 years and 118 patients, aged 9 to 93 years using a new aptt reagent (pathromtin $l). the results were compared with those obtained with a routinely used reagent (pathromtin). the reference range, factor-, heparin-and lupus anticoagulant sensitivity were determined. analysis was performed using the behring fibrintimer a (bfa) with optomechanical clot detection, the behring coagulation timer (bct) with op-"dcal clot detection by threshold and the dw test and dw confirm for lupus anticoagulant diagnostic. our results showed that the new pathromtin sl reagent met the demands for a higher factor and lupus anticoagulant sensitivity. it is highly suitable for monitoring heparin therapy and gave comparable results with the optical and the optomechanical analyser systems, hence reagent c~n be used for both systems. restenosis following percutaneous transluminal angioplasty (pta) continous to be a major clinical problem. neoinfimal hyperplasia, being the major undedying cause, can not be sufficiently avoided. vadous plasmatic coagulation and fibrinolytic factors, have been associated with artedal restenosis. anticardiolipin antibodies (act_) have been established as dsk factors for venous or arterial thrombosis. methods: in a cohort of 75 patients (53 men and 22 women, age 68±13 years) undergoing pta of a peripheral artery we prospectively evaluated whether acl could influence 6 months restenosis rate. patients were clinically examined before, 3 and 6 months after pta. noninvasive grading of artedal stenosis was done by duplex scanning of jet peak velocities. restenosis was arbitrarily defined as more than 50% occlusion of the lumen at the site of dilatation 6 months after successful intervention. laboratory investigation at the same time included acl and other known atherosklerosis risk markers, such as fibdnogen (fbg), yon willebrand factor (vwf), homocystein (hcy), c-reactive protein (crp). thrombin generation markers, such as thrombin-antithrombin iii complexes and prothrombin fragments 1+2, as well as thrombomodulin (fm) as an endothelial activation marker, were also measured. results: 31/75 (41.3%) patients were considered to have developed restenosis after 6 months. 9/75 (12%) patients were found to have positive igg-(19-35 gpl) and/or igm-acl (14-103 mpl) at all three measurements. 1/75 was negative before but seroconverted (igm) 3 months after pta. 2/10 (20%) acl-positive and 29165 (44.6%) acl-negative developed restenesis at 6 months (chi-square p-value=0.14). all above mentioned coagulation parameters did not differ between acl-positive and -negative patients, measured before or 6 months after pta. some of them are shown below (values before pta): fbg ( basilar artery stenosis is a rare event in young children. risk factors are head or neck trauma with consecutive dissection of the vertebral artery, cardiac diseases or hypercoagulability. elevated lipoprotein (a) (lp(a)) serum levels in adults can mediate atherosclerosis. in addition, lp(a) might interfere with fibrinolysis. here we report on a 3 year old boy , who presented with acute brain stem symptoms. history revealed neither trauma nor infectious disease. conventional and mr angiography showed stenosis of basilar artery without ischemic lesions. laboratory findings were normal in routine blood and csf tests. global coagulation parameters as well as procoagulant and anticoagulant factors were normal. cardiac and autoimmune disease could be ruled out. lp(a) serum levels were significantly elevated to 104 mg/dl (normal range <30 mg/dl). analysis of other family members revealed a hereditary hyperlipoproteinemia (a) which might explain family history of an increased incidence of myocardial infarction and cve in elderly family members. clinically the patient recovered completely from brain stem symptoms after heparinization and subsequent oral anticoagulation with phenprocoumon. however, radiological signs of basilar artery stenosis were progredient. in a recently developed specific test, an elevated anti-phosphatidylserin antibody titer was detected one year after primary diagnosis. in conclusion, this is the first report on a child with stenosis of the basilar artery and elevated levels of lp (a). it is unclear, whether apa contributed to the onset of basilar artery stenosis or developed secondary due to endothelial defects after thrombosis and anticoagulation. apa, however, might increase the risk of further thrombotic events in this patient. in 110 patients with thrombotic events respectively patients with systemic lupus erythematodes antioardiolipin antibodies (aca) aund lupus anticoagulant (la) were ~ea~ured. for aca detecting we use the assays from elias for igg-and ig~}-antibodies. we use as sensitive methods for detecting la in our laboratory the testkits from diagnostlca stago (staclot la with hexagonal array of phospholipids, ,ptt-la a very sensitive pttmethod and staclot p~p-a platelet neutralization procedure) and the ptt from organon teknik~ (platelin excel ls with two incubation times, 1 and 10 minutes). i"~e results of this tests were compared with three new or~e on german market: specktin apot (aktlvated plasma clotting time), specktin aptt (aptt wlth purified soy extract) and 8pecktin la (phospholipid preparation in concentrations between 10 and 500 ~g/ml); all wak chemie. traditional aptt reagents were developed for the sensitive detection of factor vib an ix as a cause of hemorhage. high sensitivity against lupus anticoagulants, which also prolong aptt, was not required for this purpose, with increasing recognition of the importance of antiphospholipid antibodies as a risk factor for thrombembolism, more sensitive reagents were designed, which now reliable detect this condition. using such reagents as a screening test in a general hospital makes it necessary to distinguish both conditions quickly. we here report an algorhythm, by which we use an inhibitor (lupus anticoagulant) sensitive (sta aptt, boehringer) and an inhibitor insensitive reagent (actin fs, dade) to distinguish anticoagulants and factor deficiencies as a cause of prolonged aptt. citrate plasma from 33 patients with various diseases showed an unexpectedly abnormal inhibitor sensitive aptt (>40s). 13 plasmas with factor deficiencies remained abnormal with the insensitive aptt reagent. a regular correction of their defect occured on mixing with normal plasma. by measurement of single coagulation factors five patients with contact factor xii deficiency were found. this condition is associated with thrombosis and very rarly with bleeding. three patients with factor xi deficiency and two patient with factor ix deficiency were also identified. antiplatelets, of any kind, permits a secondary prevention of myocard ischemic lesions. there is no general consensus regarding secondary prevention of cerebral ischemic lesions. aspirin remains the most common substance, ticlopidlne also brings about prevention, but with important secondary effects. european stroke prevention study i has demonstrated that the combination of antiplatelets, in particular aspirin/dipyridamole (persantln), is also very active. to collect more information, esps 2 was organized and 6602 patients receiving either a placebo,either 50 mg aspirin,either 400 mg sustained release form of dipyridamole (persantin (r)), or the combination aspirin/dipyrldamole, were recruited. it ended march 31st 1995 with the following conclusions: i-aspirin, 50 mg a day, brings about a significant secondary reduction of stroke (18.z %), after a two year follow-up. notwithstanding the low dose of aspirin, haemorrhages remain important. 2-dipyridamole, at 400 mg a day, brings about a significant reduction of stroke (i6.3~), similar to that of aspirin. one could thus substitute 50 mg aspirin by 400 mg dipyridamole. 3-the combination of 50 mg aspirin and 400 mg dipyridamole brings about a significantly greater reduction of stroke (37.0 ~). esps 2 revealed that a low dosage of aspirin is active, that dipyridamole alone is also active, but that the combination of both gives far better results. the study of the primary end-points,the study of the survival curves, the factorial statistical analysis and the pairwise comparison analysis, led to these conclusions. the conclusions drawn from esp£ 2 underline that the combination aspirin/dipyridamole is a privileged choice for cerebral ischemia, the state of activation of circulating platelets in acute cerebral ischemia is controversial. activation of platelets on single cell level can be assessed by determining the shape change or the expression of antigens such as p-selectin (cd62). shape change is an early and rapidly reversible event in platelet activation whereas p-selectin is irreversibly expressed on the platelet surface upon stimulation. methods: we investigated 20 untreated patients within one day after cerebral ischemia, 20 patients months after stroke treated with warfarin, and 21 age and sex matched control subjects without vascular risk factors. venous blood was collected into a fixation solution blocking the metabolic processes in platelets within milliseconds. we determined the fraction of resting discoid platelets by phase contrast microscopy. the expression of p-selectin was measured by flowcytometry. results: the fraction of platelets expressing p-selectin was higher in patients with acute cerebral ischemia (7.8_+4.7%) than in control subjects (1.9_+1.1%; p<0.001, u-test). patients with stroke (n=15, 7.8+4.5%) and patients with transient ischemic attack (tia; n=5, 7.6-+6.1%) had similar values. patients months after stroke still had higher values (4.3+9,3 %, p<0.05) than control subjects. the rate of discoid platelets was not different between patients with acute ischemia (n=17, 85.6-+8.8 %), patients months after stroke (n=19, 85.7-+5.1%) and control subjects (n=21, 86.7_+5.8 %). platelet count was not significantly different between groups. conclusion: the elevated proportion of platelets expressing pselectin indicates strong platelet activation in acute cerebral ischemia and in a majority of patients months after stroke. assessment of pselectin revealed a higher sensitivity for platelet activation after stroke or tia than analysing the reversible shape change. further studies have to clarify if monitoring of platelet activation by flowcytometry is helpful as a prognostic tool and to evaluate therapeutic strategies after stroke. vascular smooth muscle cell (smc) proliferation and migration into neointima are the hallmarks of atherogenesis. the complexity of these processes and their concerted action and interaction of molecules are yet to be fully elucidated, one crucial molecule seems to be the urokinase-type plasminogen activator receptor (upar) recently also assigned as cd87 antigen, upar serves a dual function: (1) it directs upa proteolytic activity to a special location on the cell surface and (2) induces cellular signals leading to various phenotypic changes. we have investigated the signal-transducing capacity of upar in human smcs and provide here a molecular explanation for uparrelated cellular events. activation of these cells with upa (even with inactivated catalytic center) results in the induction of tyrosine phosphorylation, suggesting modulation of upar-associated protein tyrosine kinases (ptks) upon ligand binding. we obtained patterns of tyrosine-phosphorylated proteins with molecular masses of ~ 55-65 and 35-40 kd. using antibodies against different types of ptks as well as immunoprecipitation-and immunoblotting techniques the ptks involved in the upar-signalling complex were identified to be members of the src-ptk family. the cotocalization of upar and ptks at the cell surface of smcs was further confirmed by confocal microscopy studies. we conclude that the upar-ptk complex is most likely involved in this signal transduction pathway that provides the coordinated action of extracellular proteolysis, adhesion, and cell activation, which is required for cell migration. this mechanism may be crucial for the progression of atherosclerotic plaques. activation markers of haemostasis have been found elevated in relation to diabetic vascular lesions. simultaneous pancreas-and kidney transplantation (pkt) in type i diabetes has been shown to improve diabetic complications and long term survival. we measured haemostatic vascular risk factors and activation markers in plasma of 18 patients after successful pki', 17 patients after pkt and rejection of the pancreas graft and 5 patients after pkt and rejection of the renal graft. blood samples were taken during routine ambulatory visits, patients were free of any ongoing acute disorder or transplant rejection and under continuous immunosuppressive medication. despite individually adjusted insulin therapy hba1 plasma levels increased after pancreas rejection (5,37 vs 7,i2, p<0.001). platelet counts and plasma levels of fibrinogen, f1+2 fragment, tat-, app-complex and-fibrin monomer were found significantly elevated as compared to diabetic controls but not significantly different with respect to complete or partial successful pkt. one major reason of the increased activation state of haemostasis may be cyclosporin treatment given to all patients, t-pa and pal i plasma levels were within the normal range and significantly correlated to plasma triglyceddes (r.0.049; p<0.005). d-dimer plasma levels were significantly lowered after pancreas rejection (772(375) vs 324(232) nglml; mean(sem) p<0.005), which might reflect impaired fibrin degradation related to increased glycosylation of fibrinolytic factors. in conclusion, despite the marked improvement of glucose and lipid metabolism, plasma markers of activation of coagulation and flbrinolysis are not decreased to normal after simultaneous pancreas and kidney transplantation. according to the investigations of fowler et al. and pepe et al. the probability of an ards occurring with one risk factor is 5-8%, and in the presence of several risk factors, 25%. goris et al. and johnson et al. determined the level of severity with the aid of a fixed scale: the injury severity score. all these investigations are however not to be interpreted as typical following coronary surgery. these investigations demonstrated that the kallikrein and factor xii systems are of great importance as intraoperative risk factors. here the factor xii system plays a major role with direct or indirect activation of the kauikrein-kinin system with the splitting products alpha-factor xiia and bfactor xha respectively. all ards scores take the pmn-elastase into account. if the pmn-elastase values (1000 pg/l) are constantly high postoperatively then lung complications are to be expected. patients developing an ards displayed significantly lower alpha2-macroglobulin values. patients who developed a highly significantly raised kallikrein-like acdvity (>60 u/i) after the beginning of bypass and showed constantly high values during ecc are difficult to keep under control due to the blood pressure behaviour. the platelet pal also shows a significant rise and intraoperatively runs analogous to platelet factor 4, only antiparailel, since it attacks the endothelium. we were able to show that pai-1 is suitable as an indirect marker for a possibly developing restenosis. 85% of the patients investigated with lowered pai-1 values in the postoperative phase did not develop a restenosis. however, with patients showing significantly rising pa[-1 values from the 1 st. to 3rd. postoperative day 50% of all the cases had a restenosis. a further risk factor in this respect are significantly raised fibrinogen levels which lay over 800% at the end of surgery. if these fibrinogen values do not fall from the 1st. postoperative day onwards a raised risk of thrombosis must be reckoned with in the absence of therapeutic intervention. the following parameters represented haemostaseological risk parameters with significant behaviour within the framework of this study: 1) regards the blood pressure behaviour, the kallikrein-like activity (>60 u/i); 2) with regards to the lung complications, aipha2-macroglobulin and pmn-elastase (>900 g/i); 3) and final/y as a possible marker for a developing restenosis pai-1 and fibrinogen (>800%). resulting from numerous clinical studies homocysteinemia is found to be an almost independent risk factor of atherosclerosis including thrombotic complications as well as of venous thromboembolism. experimental investigations on the underlying mechanisms suggest endothelial cell damage accompartied by the development of an atherogenic and thrombogenic potential, increased platelet reactivity, oxidative modification of ldl, and enhanced affinity of lp(a) for fibrin. to our knowledge no results are published on the influence of homoeysteine on leukoeytes although these cells are deeply involved in pathological events within the vasculature. therefore, as a first approach different functional parameters of human polymorphonuclear leukozytes (pmnl) were followed under incubation with 60, 300, and 600 i.tm (final concentration) dl-homocysteine (hc) in isolated fractions or whole blood, respectively: l) spontaneous mobility of pmnl, measured as migration distance into micropore filters in a modified boyden-chamber, is found to be significantly enhanced by the two smaller hc concentrations. 2) chemotaxis induced by 0.1 i.tm formylmethionylleueylphenylaianine (tmlp) shows no significant differences. 3)monitoring of chemiluminescence signals (autolumat lb953, berthold) is complicated as hc influences the luminol-mediated indicator reaction. adjusting appropriate conditions the following results are obtained: spontaneous chemiluminescence and that induced by zymosane, tmlp, and the ca2+-ionophore a23187 are entranced by the two higher hc concentrations. there are, however, differences between the blood donors as a minority does not respond to hc in repeated measurements. with phorbol 12-myristate 13 acetate the signal is diminished by hc in all cases and with all concentrations. 4) phagoeytosis induced by zymosane (microscopic evaluation) as well as by opsonized e. coil (cytoflowmetric evaluation) is significantly increased by the two higher hc concentrations. conclusion: the activation of human pmnl is enhanced with respect to the majority of investigated stimuli by hc in concentrations reached under pathophysiological condititions. the effect of pysical exercise on hemostatic parameters was studied in 12 patients (male, mean age: 55 [range 36-68] yrs) with angiographically documented coronary artery disease (cad) and in 11 controls (male, 53 [43-62] yrs) both participating in an 1 hour group exercise session for cardiac rehabilitation. in each group relevant arteriosclerotic lesions in carotid, abdominal and leg arteries were excluded by doppler ultrasound examinations. patients were all under 6-blocking agents and aspirin. plasma levels of prothrombin fragment 1+2 (ptfi+2) and fibrinopeptide a (fpa) reflecting formation of thrombin and fibrin, respectively, were measured at rest and immediately after 1 hour of exercise consisting of jogging, light gymnastics and ball games. training intensity in both groups was comparable as indicated by the mean heart rate during exercise corresponding in patients to 80+6% (mean-+sd) and in controls to 79-+5% of the maximal heart rate previously determined on a bicycle ergometer. baseline values for ptf1 +2 were significantly lower in oatients (0.67-+0.15 nmol/i; mean-+sd) than in controls (1.01-+0.21; p<0.001 i. after exercise we found an increase of ptf1 +2 in controls to 1.14-+0.24 nmol/i (p<0.001) while in patients ptfi+2 remained unchanged (0.67-+ 0.14 after). accordingly, exercise induced r se of fpa was more pronounced in controls (from 0.62-+0.24 to 1.60-+0.62ng/mt; p<0.001) than in patients (from 0.63-+0.26 to 1.20+0.46ng/ml; p<0.00t). we conclude that in terms of thrombin and fibrin generation exercise training does not exert detrimental effects on hemostasis in patients with cad. lower baseline values and lack of exercise induced increases of ptf1 +2 in patients with cad might be attributed to medication with aspirin and/or b-blocking agents. periodontitis marginalis (pm) is an inflammatory oral disease that is caused by gram-negative bacteria and that has a high incidence in the second half of the life. clinical signs of pm are gingival bleeding, periodontal pockets, alveolar bone destruction and loss of teeth. recent epidemiologlcal studies have provided some evidence for an association between pm and atherosclerosis. in the present paper we will summarise some of the results that we have obtained in studies on patients with pm as well as on patients with hypercholesterolaemia (hc) and atherosclerosis. pm was frequently found to be associated with hc (90 % in rapidly progressive pm) and increased reactivity of peripheral blood neutrophils and platelets (e.g. generation of oxygen radicals and paf-induced aggregation). patients with hc and atherosclerosis had a higher frequency of severe pm when compared with data on the community periodontal health. the severity of pm was higher in patients with plasma cholesterol levels _> 6.5 mm when compared to those with plasma cholesterol < 6.5 mm. in patients with coronary atherosclerosis the severity of pm was significantly correlated with plasma cholesterol level, systolic blood pressure and the number of diseased coronary arteries. these results provide further evidence for an association between pm, hc and atherosclerosis. it can be speculated that hc is not only a risk factor for atheroscterosis but also a risk factor for pm and acts by increasing the reactivity of neutrophils and platelets. on the other hand, pm as a mild chronic inflammation could promote the development of atherosclerosis due to effects of endotoxins on vessel wall, blood cells and haemostatic factors. it has been also speculated that phagocyting leukocytes in the inflamed periodontal tissues could contribute to oxidative modification of ldl. so far, there is no evidence that atherosclerosis may contribute to the pathogenesis of pro protein z (pz) is a vitamin k dependant plasma protein synthesized in the liver. it promotes the association of thrombin with phosphorlipid surfaces. recently it has been shown that a deficiency of pz may lead to a bleeding tendency. in patients undergoing chronic hemodialysis, disorders of hemostasis are common. to examine if plasma levels of pz are altered in patients with end stage renal disease we determined pz in plasma of patients at the beginning of hemodialysis treatment. the results were compared with a group of 50 healthy controls. the difference of pz levels in plasma of patients with end stage renal disease with the control group was not significant. control group was 2900 + 487 ng/ml and in patient group was 3133 + -1394 ng/ml. one patient with marked bleeding tendency after hemodialysis pz was 1387 ng/ml. we concluded that patients with bleeding disorders pz determination may be helpful. the normal range of actin fs was reinvestigated in a multicentric approach. a protocol was developed which requests from each center to assess the aptt with one common and one variable lot of actin fs in 50 samples of suspected normals. inclusion and exclusion criteria based upon the results of clotting assays, liver enzymes and clinical data were defined. results: a total of 1056 results was obtained. the majority of centers in this study used the electra 1000 or 1600 c (mla). results for the electra group (n = 6) showed a precision for the common lot of actin fs with a common lot of a three level control from 1.5 % (level 1) to 1.1% (level 3) with an excellent accuracy between the 6 centers. clotting times with the variable lots of actin fs were very similar. the results from normals, however, showed a somewhat higher dispersion using the common lot of actin fs. 4 of 6 centers had almost identical mean values (range 26.6 to 27.2 sue) whereas one reported shorter and one longer clotting times (25.2 and 29.7 sec). results with the variable lots gave almost identical results as the common one. a total of 556 results of all lots gave a normal range of 23.5 to 31.7 (5 -95 % percentiles) on electra. mean values on acl (n = 200) were 26.3, on bct, 27.65 sec, on amga coagulometric, 29.4 sec, on amga turbidimetric, 26.6 sec (n = 100 each). all centers used sarstedt monovettes with 3.2 sodium citrate. discussion: the results of this study demonstrate the lot to lot consistency of all lots of reagents included in this study since the common and variable lots showed very consistent results. interestingly in the large group of electra users the normal ranges showed some differences, though the controls in all centers were almost identical. this confirms the recommendation that a normal range as stated from the manufacturer should be used for orientation only and that each laboratory should assess its own range. direct acting anticoagulant agents such as hirudin (r-h), argatroban (arg), efegatran (efe) and peg-hirudin (ph), represent specific and potent inhibitors of thrombin. blood samples collected in r-h (10 ~g/ml), arg (50 ~tg/ml), efe (25 ~tg/ml) and ph (10 ~tg/ml) do not clot for extended periods (>24 hours), thus allowing for the collection of plasma for analytical purposes. unlike heparin, these agents do not require any plasma cofactor for their anticoagulant effect. in contrast to citrate, oxalate, edta and heparin, these antithrombin agents do not alter the electrolyte or protein composition of blood. thus, blood collected in these agents may provide a physiologically intact (native) sample for clinical laboratory profiling. we have used all of these agents to prepare whole blood and plasma samples for various diuical laboratory measuroments. plasma samples collected with these agents are obviously not suitable for global clotting tests (pt, aptt, thrombin time, fibrinogen); however, these agents are optimal anticoagulants for the collection of samples for the molecular markers of hemostatie activation, such as fibrinogen/fibrin related degradation products, prothrombin fragment, protease cleavage products, tfpi, tnf and other protein mediators. electrolytes, blood gases, enzymes and protein profiling can also be satisfactorily measured on blood samples collected with these agents. antithrombin anticoagelatad blood used fur hematologic analysis showed equivalent blood count and differential results as that obtained with edta blood. unlike other anticoagulants, these agents do not interfere in the cell staining process. washed blood cells can also be prepared using antithrombin aents supplemented buffers for morphologic and fuuctional studies. thrombin inhibitors such as hirudin have also been used for flow cytometry and image analysis of blood cells and tissue exudates. our observations suggest that these anticoagulants can be used as suitable anticoagulants for clinical laboratory blood sampling. these agents can also be used as a flush anticoagnlant fur most automated instruments as these exhibit superior anticoagulant properties to heparin. furthermore, the hematologic parameters obtained in antithrombin anticeagnlated blood may be physiologically more relevant than those determined on blood collected in edta, citrate or heparin. antithrombin ul determination is one of the most popular method for in vitro diagnostic of number of different disorders. human fhrombin a~nity purified on heparin-rnodified silica-based sorbents was used for level of antithrombine lu determination by abilgaard method in blood of 40 patients with pregnancy pathology, acute leukemia, thrombocytopenia and anemia. it was founded, that antithrombin level is decreased to 50-60% of normal values in case of pregnancy pathology, to <50% -in case of acute leukemia and thrombocytopenia, to s0% -in case of anemia. obtained results show the strong relationships between named disorders and patient antifhrombin iii level. therefore anfifhrombine iii estimation may be used as simple and quick method for preliminary diagnosis of above named disorders. bm coasys 110 is a complete automated analyzer system for coagulation tests. it is well suited for routine coagulation testing in random access in a medium throughput laboratory environment. analytical performance and practicability were tested by a common evaluation program in five hospital laboratories. within run and day to day cv's were below 5 % in different samples (controls, patients) . comparison in different therapeutic ranges confirms the declaration of the isi-value for calculating inr-values. normal values for coagulation tests with results in pdmary units were checked in 390 samples and confirmed. due to the optical measuring principle of the bm coasys 110 there was a little tendency to shorter times with the thrombin reagent. in conclusion the performance of coagulation tests with the bm coasys 110 was rated as well or better compared to existing systems in the laboratories with advantages due to short timed familiarizing and easy handling. flexibility and stability of the system permit optimal integration and innovation into the w0rkflow of the routine laboratory. the purified thrombin and antithrombin iii (at iii) have a great interest in the clinical diagnostic and treatment practice, so their isolation methods are very important. molecules of these proteins have some fragments replying for interaction with native glycosaminoglycan, heparin. this interaction is used for isolation and purification of thrombin and at ul from native materials, blood plasma or its fractional products. we have done comparative studying these proteins purification on heparin sorbenfs, which contain heparin immobilized on sificagel, modified by glycidooxipropyl, gamma-aminopropyl and tosyl chloride groups, or on cellulose: heparin-epoxy-silica (1), heparin-gammapropyl-silica (2), heparjn-tozylsilica (3), and heparin-cellulose (4). we founded that thrombin binds with all sorbents, while at iii doesn't binds with sorbents 2 and 3. there wasn't any difference between silica and cellulose sorbents in thrombin desorbfion by t m naci. at iii binds more stronger with heparin-ceuulose t[~,~n with silica sorbents but specific activity and purity degree were approximately the same on both kinds of sorbents. thrombin specific activity and purity degree were approximately twice higher on sorbents 2 and 3 in comparison with sorbents ! and 4 (2250-3000 nih units/mg versus 1260-t350 nih unlts/mg). therefore, sorbents 2 and 3 can be used for isolation and purification of thrombin and sorbents t and 4 can be used for isolation and puriiication of at ii1. we used these sorbents for large scale purification of named proteins. purified thrombin was used for production of diagnostic kits for anfithrombine iii, fibrinogen, fibrin/fibrinogen degradation products and thrombin time determination. after an aerobic or anaerobic physical exercise various alterations of the hemostatic system were detected. numerous investigations of the hemostatic system exist of running and of bicycle ergometer exercise but not of swimming. young volunteers (n=54; median age 25 years) were investigeted~ there was an aerobic exercise (achieved heart rate 130 --10/min, lactate < 4 mmol; n= 27) and an anaerobic exercise (achieved heart rate 150 ~ lo/min, lactate > 4 mmol/1; n= 27). in both groups there was a significant shortening of the ptt. under anaerobic conditions hematocrit and quick significantly increased. factor viii activity rose significantly in both groups. indicating plasmatic clotting activation there was a significant increase in molecular markers tat and f1+2 only under anaerobic conditions (tat from 2,5 to 5,4 pg/1; f1+2 from 0,87 to 0,93 nmol/1). indicating activation of fibrinolysis t-pa activity increased significantly in the anaerobic group (from 0,1 to 0,4 iu/ml) but not in the other group. this findings indicate that there is e balance in the hemostatic system by activation of clotting as well as of fibrinolytic system in young volunteers during exercise by swimming dependend on the degree of exercise load. membranes as well as compact, porous disks are successfully used for fast analytical separations of biopolymers. as far as capacity, speed and performance of separation are concerned, the supports are as effective as other recently developed fast media for the separation of biopolymers °). so far, technical difficulties have prevented the proper scaling-up of the processes and the use of membranes and compact disks for preparative separations. in this report, the use of a compact tube made of poly(glycidyl methacrylate) for fast preparative separations of proteins is shown as a possible solution of these problems. the units have yielded excellent results, regarding performance and speed of separation as well as capacity. the application of compact tubes made of poly(glycidyl methacrylate) for the preparative isolation of the coagulation factors viii and ix from human plasma shows that this method can even be used for the separation of very sensitive biopolymers. in terms of yield and purity of the isolated proteins, this method was comparable to preparative column chromatography. the period of time required for separation was five times shorter than with corresponding column chromatographical methods. our measurements showed an excellent correlation of the two systems (r=0,99). the maximum amplitudes on the roteg were on average 2.2% higher than on the hteg, corresponding to a slightly lower reverse momentum of the measuring system in comparison to the hteg. we report first results out of the evaluation of sta compact (boehringer mannheim/diagnostica stack)). sta compact is designed for automated analyses of routine and special coagulation (chronometfic, photometric [405 nm] and turbidimetric [540 run]) tests. in addition, it does measure ,,derived" fihrinogen. tests as follows were evaluated: prothrombin time (pt), partial thromboplastin time (aptt), fibrinogen (clauss method), thrombin time, at iii (chromogen), hepato quick, as well as the factors ii, v, vii, x, and viii. results: within run cvs of the clotting tests were below 2% (calculated on the basis of seconds) in most cases, day to day cvs below 4% (not measured for factors, yet). at iii yielded within run cvs below 3% in the decision range. measuring ranges: at iii: 20-140 %; fibrinogen: 1.3-9.0 g/l (plasma -dilution 1/20), after rerun with other dilutions: from 0.3 g/1 (dilution: 1/5) to 18 g[l (dilution: 1/40). method comparisons, using sta as reference, yielded slopes close to 1.00 and negligible intercepts. throughput: with routine clotting tests about 100 tests/h, in a sample selective access mode. we conclude, that sta compact allows precise measurement of routine and special coagulation tests. it is also a reliable system for photometric tests and well suited for intermediate workloads as well as stat analyses. we did evaluate ptt lt, a new liquid, silica based ptt reagent. special attention was given to reference interval and heparin sensitivity. the new reagent is well suited for the measurement of intrinsic clotting factors and is reported to have high sensitivity for lupus anticoagulants (higher sensitivity than sta aptt [boehringer mannheim = bm]). it is stable for 4 days in the cooled compartment of the sta analyzer. methods: all experiments were made on sta. for comparison, we used three other ptt reagents (a lab. routine, silica based aptt, as well as sta aptt and sta ptt kaolin from bm). in addition, thrombin time (3 u/ml thrombin, sta thrombin reagent) and heparin (chromogenic xa test, rotachrom heparin) were measured. results: within mn imprecision (n=21) was below 0.7 % cv in the normal range and in two controls (mean values: 35 s, 76 s), and 1.4 % in a heparin plasma (mean: 81 s). between day imprecision (d=10) was below 3% in two controls ( mean values: 34 s and 58 s). the upper limit of the reference range is 40 s (97.5 th perc., median: 32 s; 90 patients with normal coagulation status [routine aptt, fib., pt], median age: 54 years); almost identical reference ranges were obtained with sta aptt and the routine ptt reagent, while sta ptt kaolin showed significantly lower values (97.5th perc.: 33 s, median 28 s). method comparison study: good agreement using plasmas from patients without heparin: (y= 0a + 0.98 x, n= 198, range of(x) from 25 to 79 s, r = 0.97; x = sta aptt). the median values from 54 patients under high dose heparin were: routine ptt: 81 s, sta aptt: 75 s, ptt -lt: 82 s0 sta ptt kaolin 54 s, thrombin time 37 s and heparin 0.5 iu/ml in conclusion, results of the new reagent compare well to our routine ptt and to sta aptt system reagent. it allows sensitive monitoring of high dose heparin therapy and is well suited for detecting abnormalities of the intrinsic clotting factor pathway. is a standard technique since many years. the interpretation of the thrombelastograms has been widely based on phenomenologic observations, while there is a lack of exact information concerning the coagulation mechanisms leading to the teg amplitude (a're~). the aa'ec is a measure for the mechanical stiffness of the clot and depends on: a) fibrin formation and adequate polymerisatiun of a 3-dlmensional network: measurements with nonrecalcified citrated blood activated with adp or epinephrine (both n=10) did not show any clot formation in the teg this relies on the need for a mechanical coupling between the teg pin and cup over a distance of 1 mm, which is accomplished by the fibrin network therefore, teg can only be performed under thrombin formation and thus under thrombin-activation of the platelets in the sample. factors, which inhibit platelet aggregation but don't limit thrombin-activation of platelets, cannot be monitored by teg. b) the attachment of the dot on the surface of the teg pin and cup. according to recent literature we suggest that the attachment of the clot in the teg relies exclusively on fibrinogen/fibrin adsorption to the surfaces of the pin and cup. interruption of this attachment can result in lower amplitudes or the so-called ,,stairway" phenomenon. we could show a complete interruption of the clot attachment by dipping the pin for one second in 30% albumine solution (n=10). c) the fibrinogen concentration (fg) and platelet count (pc) of the sample. in 50 volunteers we found only a poor correlation of the maximum amplitude (ma) with fg alone (r=0.40) or pc respectively (r=0.50), while there was a very good nonlinear correlation to the product of fg and pc. we suggest that the fibrin network forms the main structure of the clot while the thrombocytes enhance its stiffness in a concentration-dependent manner. this effect of the ptatelets can be completely reversed by gplibfllla antagonists. d) adequate coagulation activation: in nonactivated teg even small amounts of inhibitors can lead to a significant reduction of the ateg. conclusion: alterations in teg measurements can be judged more properly when the underlying mechanisms are understood. the consideration of the limitations of the method allows a more specific interpretation of the results. as a response on a customer request we did investigate the sample stability of blood samples for the aptt. the study was set up in a way that simulated the conditions of a large private laboratory in which the samples arrive several hours after blood collection. blood was drawn from 10 donors into 3.2 % sodium citrate and mixed well before it was divided into several aliquots which were kept at room temperature. the aliquots were centrifuged after ~ 0,5, 11, 23 and 29 h after venopuncture and the plasma was analyzed immediately with 3 different reagents on electra 1000. results: there was a clear difference in the change of the apttover time with these reagents. also f viii (determined with a chromogenic assay with complete and standardized activation) change considerably. 2 reagent a: ellagic acid, plant phospholipid, reagent b: sulfatide/kaolin, phopholipids, reagent c: ¢llagi¢ acid, plant and rabbit brain phospholipids the increase of aptt was apparently not a function of the decrease of fviii because the in vitro f viii sensitivity of reagent b. was inferior to reagent a though reagent b showed more prolongation of the aptt than reagent a. reagent c, however, showed only minor changes in the aptt. discussion: these data show that the sample stabifity of the aptt is reagent dependent and that it is not simply a function off viii sensitivity. other factors such as the buffer system but also the sensitivitiy towards other factors than f viii seem to contribute. a comparison of the technical principle of the roteg coagulation analyser and conventional thrombelastographic systems an. calatzis, p. fritzsche. al calatzis, +m. kling, +r. hipp, a. stemberger institute for experimental surgery and +institute of anesthesiology yechnische universit~t m0nchen thrombelastography (teg) was introduced by hartert in 1948 as a method for continous registration of the coagulation process. in 1995 we presented the roteg coagulation analyser, using a newly developed technical method. in teg systems according to i/artert the sample (blood or plasma) is placed in a cup which is alternately rotated to the right and left by 4,75 °. a cylindrical pin, which is suspended freely on a torsion wire, is lowered into the blood. when coagulation starts, the clot begins to transfer the rotation of the cup to the pin against the reverse momentum of the torsion wire. the angle of the pin is electromagnetically detected, transformed to the teg amplitude and continously recorded. in the roteg the pin is attached to a short axis, which is guided by a ball beating. thus all possible movement is limited to rpotation (r_oteg). the cup is stationary, and the pin is rotated alternately by 5 ° to the right and left by a feather system. when a clot is formed, it attaches to the surfaces of the pin and cup and starts preventing their relative movements against the reverse momentum of the feather. here the reduction of the rotation of the pin, which is detected optically, is tranformed to the teg amplitude. as can be shown by theoretical analysis and by control measurements, the roteg provides the same measuring capabilities as conventional teg systems. the main advantage is the solid guiding of the measuring system, which makes the roteg easily transportable and less susceptible to shock or vibration during measurement. yhrombelastography (teg) is a standard monitoring procedure for evaluation of coagulation. usually only nonactivated native blood teg measurements (nateg) are performed, which leads to a) a long time interval until coagulation and fibrinolysis parameters are available b) very high susceptibility of the measurement to inhibitors like heparin, which disturbes the judgement of other components of coagulation, c) unspecific results. our aim was to develop a coagulation monitoring system based on teg providing fast and specific information on the different components of coagulation. methods: the following measurements are performed in paralel using disposable pins/cups (haemoscope): a) extrinsic activated teg (exteg): 3551al whole blood (wb) + 5~tl innovin (recombinant thromboplastin reagent, dade). b) intrinsic activated teg (integ): 3551al wb + 5~tl kaolin (suspension 5g/l, behring). c) aprotinin teg (apteg): exteg + 20 kie aprotinin (trasylol, bayer). d) heparinase teg (hepteg) as decribed in (1). results: exteg and integ provide information on the extrinsic/intrinsic system within 5-10 min and information on the platelet/fibrinogen status within 10-20 min. because of the addition of potent activators integ and exteg can be performed when inhibitors like heparin are present in the circulation. fibrinolysis effects can be seen on exteg and integ and by comparison of exteg and apteg (apteg: invitro-fibrinolysis inhibition by aprotinin). if fibrinolysis is detected by exteg or integ and aprotinin-susceptibility is verified by apteg, aprotinin therapy will be initiated. heparin effects are revealed by hepteg. discussion: by the comparison of parallel teg measurements which have been differently activated, specific and fast information on the different aspects of the clinical coagulation status is provided. the presented tests can be easily performed bedside and only a small specimen of whole blood is needed (0,4-1,8 ml). introduction: a severely prolonged aptt (333s; normal: 40~os) was observed during preoperative screening for a planned splenectomy in a 71-year-old man with an 8 year history of osteomyelofibrosis. fellewing neer-normal~atien (71s) of the ap'ci" after 10 rain preincubation in a kaolin based aptt assay, pk deficiency was suspected and studies were performed to further investigate the nature of the pk deficiency as well as the mechanism underlying the normalization of the prolonged aptt by increasing the preincubation time. methods: the apl-r assay was peal'armed using kaolin/inesithin. high molecular weight kininogen clotting activitiy (hk:c), fxii:c end pk:c were measured by an aptt based assay using neothromtin ® (behnng) and 1 rain (pk:c) or 4 min (hk:c, fxli:c) preincubation. pk amidolytic activity (pk:am) was assayed using cosset pk ~ (chromogenix) and pk antigen (pk:ag) by quantitative immunoblotting. fxll and hk proteolysis dunng activation of plasma by kaolin (10mg/ml at 37=c) or ds (12.5~tglml at 4=c) was demonstrated by immunotilotting assays of fxii and hk following sds-page. assay pk:c pk:am pk:a~i fxfi: the propositus had pk:c<5%, pk:am=5% and pi~ag<2.5% as compared to normal pooled human p(asma (nhp). his son and two daughters had pk:c-50% and normal aptt values, incubation of the propositus' plasma with ds did not result in fxii or hk cleavage within 180 rain, whereas jn nhp detectable f×ii and hk proteolysis occurred after 5 rain and complete proteolysis was observed after -120 rain. in contrast, kaolin activation of propositus' plasma led to slow activation of fxii after 10 rain, presumably by autoactivation, and to fxlla-induced hk proteolysis. near-normalization of the propositus' aptt by prolongation of the preincubation time paralleled fxii autoactivation as evidenced by immunobletting. we describe a propositus with severely prolonged aptt due to hereditary, crm negative pk deficiency suffering from omf. activation with a particulate suspension of kaolin led to slow fxii autoactivation and hk proteolysis, whereas ds in solution did not induce fxii or hk cleavage. fxii autoactivation seems to be responsible for the normalization of the prolonged aptt in pk deficiency after prolonged preincubation times. in our study we compared a conventional bag with silicone tubing (a) for blood donation with 2 new ones (]3 from biotrans and c from baxter) with a newly developed y-shaped adapter. this adapter is integrated into the tubing and therefore provides the advantage for drawing blood samples in a closed system. the 3 systems were identical in amount and content of anticoagulant, i. e. 63 ml of cpd per bag resulting in approximately 14% of the final whole blood volume. the purpose of the study was to determine whether the different tubings can influence the quality of plasma products conceming the blood coagulation system. in plasma samples we measured several factors of the procoagnlatory and fibrinolytic systems. intralndividual control eitrated (.135 m) blood samples were initially drawn from the contralateral cubital vein from the same male donor (34 in each group). in all bag samples we found small but significantly higher levels of the global test parameters ap'it and ti" compared to controls, indicating a higher amount of anticoagulant. pt, however, revealed no differences, thus suggesting that factor activities were not altered (statistics according to mann-whimey). increase of procoagulatory activity measured as tat complexes showed elevated levels in bags a and c whereas prothrombin fragments fl+2 decreased only in a. conceming the fibrinolytic system, plasminogen a~tivators and pai-1 values were diminished in all three systems 03 < a < c) compared to controls. d-dimers were lowest in a followed by slightly higher values in c, controls and b. fibrin monomers did not reveal any significant differences: a < c < controls < b. in summary, the quality, of the 3 different blood sampling devices was comparable to the intraindividual controls as to factor activities measured by global tests. the activation of the procoagulatory and fibrinotytic systems was slightly but in most cases significantly higher in the two new devices than in the conventional one. all values, however, obtained from the plasma samples did not exceed the normal range of healthy blood donors. therefore we concluded that the two new closed blood drawing systems are favorable for blood donating procedures. in 20 patients with acute myocardial infarction (ami) and thromholytie therapy (13 patients with rt-pa, 6 patients with streptokinase and one with heparln) with ck, myoglobin and ekg criterions the patients were divided in two groups (reperfusion/no fellow two hours after starting the thrombolytic therapy) . blood samples were taken before, 30 rain, i h, 2 h, 4 h, 8 h,12 h after lysis and than every day till day 10. because of the central role of factor xii in activation of coagulation, fibrinolysis, kallikreln-kinin-system and complement cascade we investlgate the role of factor xila initiated by ami and the relation of factor xiia to the thrombolytie agent and reoeclusion rate. for the investigatlens we take the kits from shield diagnostics (xiia), behring diagnostica (c~-inactivator, pl~nogen, ~-antip]~n~n, pap), chromogefiilx ab (prekallikrein) and di~nostica stage (vile). the results: there is an increase of factor xiia immediately after starting the fihrinolysis (max. 30 rain after starting); the increase 5/i independently of the thrombolytie agent. parallel to factor xiia raises factor viia without significant changes of c1-1naotor and prekalllkrein. that means: activation of xiia and fibrinolytic pathway leads to relatively mild c.hanges in kallikrein system, hut to significant activation of extrinsic system by vila-tissue factor. in some patients is an additional rise in the system xiia -viia, when the fibrinolytic system is already in the normal range. there will need further investigations to define the risk of reocclusion as a result of activation of faktor viia by faktor >li ia. autoimmune thrombocytopenic purpura (aitp) is a frequent complication of chronic lymphocytic leukemia (cll] which developes on different stages of the disease and needs special treatment measure. mechanism of autoimmune disorders in cll remains uncleared. we investigated immunologic phenotype of blood lymphoid cells in 22 patients suffering from cll with aitp. in these patients we did not observe disorders in expression of b-lineage markers as compared with cll patients without immune complications (13 patients). but in the 1st group of the patients the greater number of b-celts expressed markers of activation. according to ig heavy chain expression, the lymphocytes in most cases of cll complicated by aitp had more mature phenotype. in all patients with k phenofype of cll lymphocytes we found immune disorders. the development of aitp was accompanied by lowered level of t cells and changed dis'flibution of their immunoreguiatory subsets: diminished number of cdz~cells and increased one of cd~'÷lymphocytes. the results of our investigations undirectly proved that malignant b-cells in cll are involved in production of autoantibodies against blood cells. dysbalance in t-cell system with functional disturbances of immunoregulation are significant in development of autoimmune complications in cll a24 in women with severe fvii deficency (<10%) hypermenorrhagia may cause life threatening blood loss. therefore, hysterectomy at a young age is reported frequently in the literature. a 12 year old girl without history for a bleeding disorder was transfered with hypermenorrhagia. the initial laboratory data revealed an abnormal quick-test of 30% due to fvll of 9,0%, normal platelet count and hemoglobin level of 7,2 g/dl. antifibrinolytic therapy (tranexamic acid 4x15mg/kg bw/d) and lynestrenol substitution were started to reduce the hemorrrhage. despite treatment the daily blood loss increased to a maximum of 290ml. therefore, substitution therapy with recombinant fvila (rfvila) (novonordisk) was started at a dose of 15 ilg/kg bw q 6 h. subsequently blood loss decreased to 30ml/d, but even with an increasing dose of rfvlla up to 35 i~g/kg bwq 4h (fvil activity max. 7400% 10 min after injection) and additional hormonal support with a lh-fsh-anatgonist some hemorrhage remained. a short .course of methergin was stopped due to severe pain. ultrasound of the uterus revealed a hypertrophic endometrium causing the persistent bleeding. it decreased slowly over several weeks and hemorrhage stopped completely after 40 d. the total rfvlla dose administered was 118 rag. no side effects were observed. no transfusions of blood products were necessary. currently, menstrual cycle is suppressed by estriosuccinate. conclusion: due to close cooperation with a specialised gynecologist, hypermenorrhagia was controlled and in this woman with severe fvll deficiency hysterectomy was avoided. in three male members aged between 27 and 52 years of a family suffering from inherited bleeding disorders the diagnosis of protein z deficiency was established. plasma protein z evaluated by elisa (asserachrom protein z, diagnostica stago, france) ranged between 200 and 300 ng/ml. the patients mostly suffered from moderate bleeding complications like prolonged bleeding secondary to trauma or invasive measures and also spontaneous hematuria. previous laboratory investigations revealed variable platelet function deficiencies and transitory boderline decrease of von-willebrand factor. spontaneous bleedings were rarely recognized, however, they occured more frequently when analgetics were taken. bleeding complications showed good response to hemostyptic measures and antifibrinolytic therapy. the use of pcc containing a high level of protein z in these patients is restrained to severe bleeding disorders or major surgery. defibrotide is a mammalian polydeoxyribonucleotide derived anti-ischemic and antithrombotic drug (crinos s.p.a., v"flla guardia, italy). while the drug is known to produce polytherapeutic effects owing to its multicomponent nature, the exact mechanisms of its anti-ischemic effects remain unknown at this time. since defibrotide is found to be effective in ischemic disorders such as paod, vod related occlusive disorders and related rnicroangiopathic conditions, we studied the effect of this drug on the contraction of dog and pig arterial strip/rings obtained from various sites. in vitro supplementation ofdefibrotide to the organ bath containing control dog and pig arterial rings did not modulate the serotonin and thromboxane (generated) contraction, however, tissues obtained from dogs treated with 10 mg/kg defibrotide iv exhibited a profound desensitization to the agonist induced contractile process. the time course of these effects was found to be much larger than the plasma half-life of defibrotide. this presentation will provide additional data on the effect of defibrotide on the contraction of vascular smooth muscles as a possible explanation for the anti-ischemic effects of defibrotide. a. wehmeier, a. popescu, w. schneider klinik for h,~matologie, onkologie und klinische immunologie der heinrich-heine-universit&t d0sseldorf in chronic myeloid leukemia (cml), evolution of blast crisis is the limiting factor of survival. however, as in other chronic myeloproliferative disorders, bleeding and thrombotic complications are a major source of morbidity but their incidence has rarely been analysed in larger patient groups. we retrospectively evaluated 182 patients with cml during chronic phase (170 cases), accelerated disease (58 cases), and blast cdsis (72 cases), and determined the incidence of thrombohemorrhagic complications in relation to the stage of the disease. in chronic phase, 28 patients had bleeding complications (8.4%/patient year) and 15 patients thrombotic episodes (4%/patient year). the incidence of bleeding increased significantly in accelerated disease (18 patients, 51.2%/patient year) and blast crisis (37 patients, 347%/patient year), and many patients had repeated complications. contrary to our expectations, the incidence of thrombotic complications also increased to 10.2%/patient year in accelerated phase and 39.8% /patient year in blast crisis, tn chronic phase, 3 patients died because of bleeding events. in accelerated phase, 5 patients died due to bleeding and 1 patient due to thrombotic complications. in blast crisis, bleeding was associated with 21 deaths, and pulmonary embolism with 2 deaths. analysis of the cause of thrombohemorrhagic complications revealed that in chronic phase, bleeding was often associated with uncontrolled busutfan therapy, whereas in blast crisis, severe bleeding occurred mainly when platelet counts were low and peripheral blasts increased. however, there was no obvious explanation for thrombotic complications. we conclude that bleeding and thrombotic complications are a major source of morbidity and mortality also in cml, and that the incidence of such complications increase in advanced stages of the disease. klinik for innere medizin °, klinikum schwerin patients suffering from primary or secondary amyloidosis may occasionally acquire a coagulation disorder characterised by isolated factor x deficiency. we report on a 60-years-old man who presented with lower gastrointestinal bleeding and prolonged prothrombin time (quick 50 %). amyloidosis was suspected and proven using biopsy of the rectum and histological analysis. in addition, a monoclonal gammopathy of undetermined significance was diagnosed by immunofixation (light chain, type x). detailed investigation of the prolonged prothrombin time led to the discovery of a pronounced factor x deficiency (residual activity 4 %). inhibitors of coagulation factors could not be demonstrated. the treatment of the patient consisted of red blood cell transfusion and infusion of prothrombin complex concentrates. due to the extremely rapid clearance of infused factor x, no increase of its activity was observed. chemotherapy of the monoclonal gammopathy was initiated (melphalan/ prednisone). over the following six months the frequency of major bleeding episodes gradually decreased. however, subclinical occult bleeding continued. the factor x activity was repeatedly found between 10 and 12 %. we support the suggestion from literature data that clinically relevant bleeding episodes are likely to occur in patients with amyloidosis-associated factor x deficiency if the residual activity is below 10 %. sepsis and septic shock is a disease entity which is characterized by inflammatory reactions (sirs), coagulation abnormalities (dic), organ failure (mof) and severe hemodynamic alteration frequently leading to death in a shock. the aim of our studies was to investigate the efficacy of antithrombin iii (kybernin ®) on ~he outcome of septic shock in a pig endotoxemic model. pigs, in this model respond to lps with elevated tnflevels, decreased leukocytes and platelets counts, increased tat and fibrin monomer levels, hypotension and in increase of the pulmonary arterial pressure (pap), indicating impaired lung function. a total number of 13 male castrated juvenile domestic pigs (25 -30 kg) were anaesthetized, ventilated mechanically and infused with saimonella abortus equi lipopolysaccharide (s. equ-lps) over three hours (0.5 ~g/kg * h). a swan-ganz-catheter was inserted into the pulmonary artery to measure the pap. animals were allocated to two groups,, the treatment group (n = 7) received antithrombin iii (at iii) according to the following regimen: 250 u/kg (t = 60 -30, i. v. infusion), 125 u/kg (i. v. bolus, t = 0) and 250 u/kg (t = 180 -240 rain, i. v. infusion). the placebo group ( n = 6) received the appropriate amount of human serum albumin: 50 -25 -50 mg/kg (same schedule as with at iii). main objective was defined as the mortality rate at six hours a_~er s. equ-lps infusion. whereas in the placebo group 4 out of 6 animal died (mortality rate: 66 %) all at iii-treated pigs survived the observation period of 6 hours (p < 0.05, x2-test). the at iii group was shown to have a lower pap than the control group, especially the second peak of hypertension was abolished by at iii. it is therefore concluded that at iii should be a useful tool for the treatment of severe sepsis and septic shock. in a nationwide monthly survey all childrens hospitals in germany (esped) were asked to clinical and therapeutical informations about children suffering from pmi. during july 94 till june 95 299 children were registered. from these, 87 had either ecchymoses and/or necroses related to an increased mordibity and mortality (20%), whereas 212 showed no bleeding signs except for petechiae. of these children one died. the therapeutic interventions concerning hemostasis are listed according to the defined two risk ~oups. from the patients with ecchymoses or necroses, 13/87 received combination therapy (compared to 5/212 with petechiae or no bleeding sign) of at iii, heparin and/or plasma. only t child received protein c concentrate. the data show that children with low risk did in part receive higher doses of heparin and/or at iii concentrate than did high risk patients, whereas plasma therapy was adjusted to severity of eoagnlopathy. furthermore, the wide range of given therapeutics allows no information about the different medications. therefore, controlled studies with respect to the different therapeutic interventions in children with high risk pmi is desirable. a fully automated procedure for the reptilase time assay y. schmitt (1) and h.j. kolde (2) (1) institute for laboratory medicine, st~dtisches klinikum, darmstadt, frg, (2) dade diagnostics, unterschlei6heim the reptilase time assay is a relatively simple technique for the detection of fibrinogen degradation products and fibrinogen deficiency or abnormality. the procedure is performed with citrated plasma and batroxobin reagent, a snake venom enzyme from bothrops atrox. this enzyme cleaves fibrinogen by releasing fibdno peptide a only but not fibdno peptide b. in contrast to the physiological enzyme thrombin that is readily neutralized by antithrombin iii and hepadn batroxobin is not inactivated by physiological inhibitors. at present this assay is mainly performed manually or on mechanical instruments. we have adapted this assay to the electra 1000 fully automated coagulation analyzer (medical laboratory automation, pleasantville, n.y.) using the thrombin clotting time procedure in the instrument software with batroxobin reagent (dade diathe clot formation is registrated turbidimetrically and the dotting time is pdnted. the within run precision (n= 10) of this procedure was tested with two plasmas from the daily routine and was between 2.8 and 3.4 %. in 25 normal samples we found clotting times from 10.5 to 12.8 sec. in 30 samples with liver disease (confirmed by pseudochlinesterase < 2000 u/ml) or on thrombolysis therapy with streptokinase or urokinase the fully automated assay on the electra was compared to the semiautomatic method using a kc 10 coagulometer (amelung, lemgo, germany) based on a rolling metal ball pdnciple and magnetic endpoint detection. the two assays agreed very well with a correlation coefficient of r = 0,948 and a regression line according to passing and bablok of y = 1.0 x + 1.7. these data show that the reptilase time can be performed with good precision and with good correlation to the manual technique on mechanical instruments on the electra 1000. introduction: disseminated intravasal coagulation (dic), due to a massive activation of the coagulation system, is frequently observed in intensive care patients suffering from severe underlying diseases. laboratory diagnosis of dic is based on different coagulation tests, but unfortunately the routine haemostaseological parameters react with latency in the course of acute dic objective: in four cases from a cohort of 43 patients with severe sepsis and dic we analysed special haemostaseological parameters (tat, f1-t2, d-dimers, human-leucocyte-elastese (file), catepsin g and heparin cofactor ii (hc ii)) and correlated them with a mof-score in order to test their predictability on the prognosis of these patients. results: all patients were substituted with at iii concentrate. l,1 the investigated patients median time of treatment with at iii concentrate was 8 (6-9) days and median time of dic-duration was 6 (4-8) days. none of the presented patients died during observation period. all analysed parameters, except d-dimers, showed a sufficient correlation with the evaluated mof-score (tat: r= 0,78; f1-f2: r= 0,84; hle: r= 0,71; catepsin g: r=-0,75; hc ii: 1"=-0,88). the d-dimers did not correlate with the mof-score, which is probably due to the delayed reactive hyperfibrinolysis in the course of dic. furthermore, the decrease of the tat-complexes, f1-f2, hle and catepsin g levels were followed by an increase of at hi and hc ii activity. conclusion: in general the analysed activation markers and coagulation parameters are sufficiently to describe the ongoing process of the dic. the hyperfibrinolytic activity of dic is sufficiently represented by the d-dimer test, but is of defered reactivity in the course of dic. unfortunately these parameters are not established in the routine monitoring of dic on intensive care units and therefore further studies are needed to investigate the practicability and reliability in the daily routine monitoring. we have previously reported that notoginsenoside r1 (ng-r1) has an effect on counteracting lipopolysaccharide (lps) induced upregulation of plasminogen activator inhibitor-1 and tissue factor expression in cultured human umbilical vein endothelial ceils in vitro and in mice in vivo [fibrinolysis 1994;8:(suppl 1)119]. in this study we investigated the effect of ng-r1 on prevention of lps induced lethal toxicity in mice. because mice are relatively resistant to lps when applied as a single agent, we sensitized them by simultaneous treatment with d-galactosamlne. the 80% lethality induced by lps (1.5 mg/mouse) plus d-galactosamine (8 mg/mouse) in c3hs-ie mice was reduced to 16% by simultaneous administration of ng-r1 (1.5 mg/mouse) with lps/galactosamine (p<0.05 by x 2 test). ng-r1 also significantly delayed lps/galactosamine induced lethal toxicity from 12 hours to 30 hours with all animals surviving beyond 30 hours. because lethality induced by lps involves the synergistic effect of multiple effector molecules such as tumor necrosis factor (tnf)-ct, interleukin (il)-i, interferon 3' etc., we also investigated the effect of ng-r1 on lps induced tnf-ct production from leukocytes in cultured human whole blood cells (hwbcs) ex vivo. the production of tnf--ct induced by lps (1 ng/ml for 24 hours) in the supernatant of hwbcs was inhibited by 46% and 22% respectively, when the cells were incubated 1 ng/ml or 10 ng/ml lps together with 100 i~g/ml ng-r1, respectively (tnf-ct concentration, 1 ng/ml lps treated cells: 297+192 pg/ml, i ng/ml lps plus 100 l.tg/ml ng-ri treated cells: 162+137 pg/ml, p<0.01; 10 ng/ml lps treated cells: 3094_+487 pg/ml, 10 ng/ml lps plus 100 pg/ml ng-r1 treated cells 2423+713 pg/ml, /'=-0.02). the present results suggest that ng-r1 can prevent the onset of lps toxicity as well as the lps induction of cytokines. therefor ng-ri may be effective in preventing the effects of septic shock in gram-negative infections. to elucidate the mechanisms by which coagulation is initiated in septic patients in vivo, coagulation measurements were prospectively evaluated in patients with severe chemotherapyinduced neutropenia. this group of patients was chosen because of their high risk of developing severe septic complications, thus allowing serial prospective coagulation testing prior to and during evolving sepsis or septic shock. 62 patients with febrile infectious events were accrued to the study. of these, 13 patients progressed to severe sepsis and an additional 13 patients to septic shock. at onset of fever, factor (f) vlla activity, f vii antigen and antithrombin iii (at iii) activity decreased from normal baseline revels and were significantly lower in the group of patients who progressed to septic shock compared to those that developed severe sepsis (medians: 0.3 versus 1.4 ng/ml, 21 versus 86 u/dl and 45 versus 95%; p < 0.001 ). the decrease of these variables in septic shock was accompanied by an increase in a marker of thrombin generation like prothrombin fragment 1 + 2 (medians: 3.6 versus 1.4 rim; p=o.05). these differences were sustained throughout the septic episode (p < 0.0001 ). f vlla and at ill levels of <0.8 ng/ml and <70%, respectively, at onset of fever predicted a lethal outcome with a sensitivity of 100 and 85%, and a specificity of 75 and 85%, respectively. in contrast, fxila-alpha antigen levels were not different between both groups at onset of fever and were only marginally higher further during the course of septic shock (p=o.001). thus, septic shock in neutropenia is associated with significant coagulation activation, presumably driven by the tissue factor pathway rather than the contact system. furthermore, in septicemia both f vlla and at iii measurements are sensitive markers of an unfavourable prognosis. hemostatic parameters in sepsis patients treated with anti-tnfct monoclonal antibodies c. salat 1, p. boekstegers 2, e. holler 1,3, b. reinhardt i, r. pihusch 1, k. werdan 2, m. kaul 4, t. beinert 2, e. hiller 1 med. klinik iii 1 und i 2, klinikum grosshadern der ludwig-maximilians-universitat monchen, h~imatologikum der gsf 3, knoll ag ludwigshafen 4 tumor necrosis factor et (tnfc~) is a central mediator in the pathogenesis of sepsis and septic shock. as administration of anti-tnfct monoclonal antibodies was able to protect animals from an otherwise lethal endotoxin challenge clinical studies were initiated in patients with sepis. tnfct exerts a procoagulant effect, e.g. by enhancing pai-i and activating thrombin as indicated by an increase in tat and pf 1/2 levels. therefore it may be involved in disseminated intravascular coagulation in sepsis. we determined tat, pf 1/2, d-dimers, tpa, upa, pai-i and vwf levels in 30 patients with sepsis or septic shock. 14 patients received the anti-tnfa monoclonal antibody mak 195f (knoll ag, ludwigshafen), whereas 16 patients served as controls. we found a significantly lower level ofupa in anti-tnfc~ treated patients. since the difference existed before onset of treatment it can not be attributed to tnfot antagonisation. all other parameters investigated did not differ significantly between the two groups throughout the study period. failure to detect modulation of hemostasis by anti-tnf~ might be explained by delayed initiation of treatment in clinical sepsis. in animal experiments it has been observed that the antibody prevented lethal endotoxin effects when given prophylactically or 30 minutes after endotoxin challenge, but not when it was administered 2.5 hours later. in addition, beneficial clinical and hemostatic effects of tnfet antagonisation might be observed only in subgroups of patients with hyperinflammatory sepsis. larger studies addressing this point are under way. protease receptors for thrombin and trypsin have been described for different cell lines. we investigated the ability of trypsin to activate human umbilical vein endothelial cells (huvec). cell activation was measured by the increase of intracellular free ca 2* (caff) with help of microscope fiuorometry (fura-2) and by the von willebrand factor release measured by a sandwich elisa. incubation of huvec with thrombin (1u/ml) or trypsin (10nm) showed a 2-10 fold increase of c~ff. a subsequent homologous stimulation after 80 s lead to a 2-5 fold lower concentration of ca~ 2÷ compared to the first stimulation. therefore cells have been desensitised by the first stimulation. inhibition of the proteolytical activity of trypsin by soybean trypsin inhibitor was followed by failure of trypsin inducing an increase of ca~ 2÷ concentration. in cross stimulation experiments with thrombin and trypsin, we could demonstrate, that cells first stimulated with thrombin showed a second maximal response by subsequent stimulation with trypsin. the same effect was measured with first stimulus trypsin and second stimulus thrombin. trypsin and thrombin induced a release of von willebrand factor (2-5 fold in comparison to unstimulated cells). we found a vwf release dependent on the concentration of trypsin similar to thrombin. an electrophoretic analysis of the released von willebrand factor showed a different multimeric composition of vwf between trypsin and thrombin stimulation. these results indicate, that there might be a protease receptor on huvec for trypsin being different from the thrombin receptor. clinical and laboratory findings of coagulopathy were investigated by an 1-year-survey to 320 children's hospitals. 291 meningococcal infections were evaluable. severe disease (characterized by need for mechanical ventilation, dialysis and/or catecholamines) was seen in 42 of these children; 29 of those survived and 13 died. clinical signs of severe coagulopathy were seen in 83 children: ecchymoses (n = 73) and skin necrosis (n = 36) were associated with increased mortality (16% and 20%, resp., compared to 4.5% overall mortality). five of 29 surviving children with skin necroses required surgical interventions (skin transplantation and/or amputations). petechiae were frequent (n = 156) and as isolated finding not related to severe disease or fatal outcome (6% mortaliy). platelet counts at admission were lower in non-survivors (10th-90th percentile: 30 -450.000/gl, median: 139.000/i.tl) than in survivors (10th-90th percentile: 140 -480.000/i.tl, median: 242.000/gl). at iii values showed no difference between survivors and non-survivors. protein c was available in few patients (n =14): in this subgroup, protein c was lowered in patients with limited disease (10th-90th percentile: 20 -105%, median: 48%) as well as severe disease (10th-90th percentile: 30 -75%, median: 60%). in conclusion, the findings "ecehymoses" and "skin necroses" were related to fatal outcome and therefore included in a prognostic score for severity of meningncoccal disease. the influence of irradiation on pai-i and vwf levels in human umbilical vein endothelial cell cultures k. fragiadaki, c. salat, r. pihusch, b. reinhardt, m penovici, e. hiller med. klinik iii, klinikum grosshadern der ludwig-maximilians-universitat monchen an elevation of pai-1 in bone marrow transplant recipients developing veno-occlusive disease (vod) of the liver has been described earlier. endothelial cell damage due to the preparative myeloablative radioehemotherapy is supposed to be an important step in the pathogenesis of the disease, which is characterized by an obstruction of small intrahepatic venules. in order to investigate a possible role of irradiation we studied the influence of several doses (0, 5, 15, 30 gy) on pai-1 and vwf levels in the supematant of human umbilical vein endothelial cell cultures (huvec). pai-1 antigen and vwf were determined by enzyme immunoassays. whereas pai-1 and vwf levels remained unchanged alter irradiation with 5 gy and in control cultures, a rise was observed one day after irradiation with 15 gy (mean day 0"-)day +1) in pai-1 (100,0% --)171,2 %) and vwf (100%--)159,7%) levels. the increase was more pronounced and reached levels of statistical significance after a dose of 30 cry (pai-1 100%--) 278,7% and vwf 100%--)168%). both pai-1 and vwf levels decreased on day 2 after irradiation with 15 and 30 gy. our results indicate that irradiation induces an increase of pal-1 and vwf in endothelial cells. nevertheless, this effect was observed only in doses above those ones used during conditioning when patients receive 3x4 gy. additional factors seem to be of significance. cytokines like tnfo~ enhance pai-1 and vwf in endothelial cell cultures and are known to be elevated in bmt-associated complications. it can be speculated that irradiation in concert with these factors may contribute to the development of veno-occlusive disease. disseminated intravascular coagulation is characterized by high consumption of coagulation factors, systemic elevation of fibrinolysis by tpa and concomitant elevation of pai-i secreted from inflamed endothelial cells. in an attempt to investigate the contribution of inflammatory cytokines, endothelial cells lines of microvascular origin were stimulated in vitro and pal-1 antigen was measured 2h, 4h and 24h after stimulation. in contrast to results published from experiments performed with macrovascular human umbilical vein cells (huve), our results obtained with 3 different microvascular endothelia isolated from skin, solid tumor tissue and bone marrow revealed that inflammatory cytokines reduced pal-1 antigen levels. in addition to tnf-a (25ng/ml) and lps (10pg/ml), we found that il-10 (100 u/ml) and gm-csf (100 u/rot) also reduced pai-i levels within the first 2h of incubation (from 120ng/ml to 80-110 ng/mll and the effect was even more pronounced after 4h and 24h (from 380 ng/ml to 250 ng/ml). il-1 (10 u/ml) and lps (10 pg/l) also reduced constitutive levels of pal-1 but the effect occured later than 4h after addition of the stimulator. the strongest synergistic effect was demonstrated with gm-csf plus il-1 resulting in pal-1 suppression of 50% after 2h and 30% after 24h. in contrast, g-csf (300 u/ml) induced the immediate (120 to 140 ng/ml after 2h and 380 to 420 ng/ml after 24h) upregulation of pal-1 antigen. stimulation of pat-1 levels was also observed with tgf-i~ (10 pg/ml), however not earlier than 18h of incubation. interestingly, both stimulatory cytokines, ie. g-csf and tgf-13, alone were able to counteract the decrease of pat-1 antigen by tnf-a but only a combination of g-csf plus tgf-g neutralized the effect by il-1. results indicate that inflammatory cytokines regulate pal-1 fibrinolysis in a synergistic and antagonistic fashion. we established the culture of human brain microvascular endothelial cells (hbmec) in order to investigate the pathophysiology of hu~man cerebral malada, which is still associated with a high mortality rate. it is widely accepted that among the reasons for the fatal outcome of cerebral malaria, the interaction of endothelial cells with cytokines and paras lites with subsequent changes in haemostaseological parameters is involved. the human microvascular endothelium may therefore play a deci §ive role in the pathophysiology of cerebral malaria. ery throcytes containing later stages of p. falciparum specifically bind to capillary ec in vivo (sequestration). tnf-cq il-1 and il-6 are considerably elevated in severe malaria. coagulation factors such as tissue factor and von willebrand factor are affected by malada suggesting the involvement of the hbmec in cerebral malada. so far, research on the involvement of the hbmec has been performed on ec cultured from human umblilical veins (huvec). the relevance of this model may be questioned on t, ,he grounds that the capillary endothelium probably plays a greater role than the endothelium of the large vessels. besides, some propertie.$ of the endothelioum seem to vary, upon the organ of origi/n. for the~ reasons, our laboratory has established the hbmec as a model to study the pathophysiology of human cerebral malaria. to demonstrate the relevance of this model in the context of malaria, hbmec were challenged with sera from different patients with severe p. falciparum malaria and with serum from a healthy donor. we can demonstrate that in cells challenged with malaria patient sera icam-1 and substance p were upregulated. on the other hand cells challenged with serum from a healthy donor expressed neither icam-1 nor substance p. these results strongly suggest the relevance of this model for vessel involvement in malaria. both, histamine and serotonin have been described as potent stimulators of yon willebrand factor (vwf) release from human umbilical vein endothelial cells (huvec). we performed experiments to differentiate the receptors for histamine and serotonin induced vwf release. absolutely unexpected we don't found any significant vwf release after the addition of serotonin to huvec or human artery endothelial cells (huaec) in concentrations from 0.1 ijm to 50 pm. in the case of histamine (0.1 pm -50 pm) we measured a vwf release 2-5 fold compared to unstimulated cells. this release was in the same order of magnitude as the release induced with 11u thrombin. to verify these results we measured the effect of histamine and serotonin on the intracellular ca 2÷ concentration (ca~ 2÷) in huvec and huaec. cells were labelled with fura-2 and the change in fluorescence after agonist addition was measured with a microscope fluorometer. using the same agonist concentrations as above we found an 5-10 fold increase of caj 2. with histamine or thrombin but no effect by addition of serotonin. this results indicate a similar activation of human endothelial cells by histamine and thrombin and that serotonin don't stimulate endothelial vwf release or increase of cay. activation and/or dysfunction of the endothelium can be triggered by cytokines (e.g. interleukin-2, tumor necrosis factor-alpha) or bacterial substances (e.g. endotoxins) and may contribute to shock and multi organ failure. pal-l and tm were assessed as parameters of activated endothelium following bsct in three to four days intervals from start of conditioning therapy through day +35. data were compared to the occurrence of sepsis, veno-occlusive disease (vod), capillary leakage syndrome (cls) and graftversus-host-disease (gvhd). patients with neither complication served as controls. no *days after stem cell tranplantation pai-1 and tm were increased in all patients with sepsis, cls~ vod and/or gvhd. pai-1 peaked at days 14 to 18 and the increase was highest in sepsis and lowest in cls. the increase in tm values was somewhat delayed (day +24) and was highest in vod and cls and lowest in gvhd. pai-1 and tm are sensitive markers of endothelial activation in sepsis, vod, cls, and/or gvhd, but they do not allow a differention between these complications. endothelin (et) is the most potent vasoconstrictor. it is known that et plasma concentration is correlated with a poor prognosis in patients with non ischemic cardiomyopathy (cm). the contribution of the heart to the production of et is still unknown. to investigate the pathogenetic mechanism in patients without coronary artery disease (cad), we examined 13 patients with hypertension ( . pulmonary capillary wedge pressure (pcwp) was measured in all patients. et and its precursor big-endothelin (bet) were determined at rest and after pharmacological stimulation with dipyridamole (0.5 mg/kg body weight), that increases coronary blood flow by factor 2 -4 on a non endothelial pathway. cardiac coronary et and bet concentrations were determined from the arterial blood samples, obtained from the aorta, and simultaneously from the coronary sinus (venous blood). blood samples were collected into ice chilled vacutainer tubes and stored after centrifugation at -70 *c. et and bet were analysed after extraction by a sepal< c 18 cartridge by radio immuno assay technique (immundiagnostik). it is concluded that et is increased with elevated filling pressures of the heart in patients with cm. it is not produced in considerable quantity by the heart neither at rest nor at increased blood flow. there4ore the lung has to be considered as the major organ for the production of et and bet in patients without cad. to characterize the incompatibility of blood with foreign surfaces valide in vitro methods especially in testing of platelet function are neceessary. it seems to be effective to use test systems which can also be helpful lateron in the clinic when foreign surfaces (e.g. venous catheters) are used and evaluated in so called phase-4-studies. we studied the influence of 21 reference polymers under standardized and controlled flow conditions on platelets in citrated blood specimen of healthy blood donors.the following tests were performed pre and post platelet-pol)aner contact: decrease of platelet count, platelet aggregation (wu-gmtemeyer index), analysis of platelet spreading capacity on standardized plastic surfaces by using a visual microscopic evaluation according to breddin and bfirck (1963) and an interactive computer-aided system (ibas, kontron gmbh, manchen, frg) by digitalizing the morphological picture of the platelet slides and area detection with a resolution of 512x512 pixels. results: platelet counts showed significant differences pre and post polymer contact, the wu-grotemeyer index demonstrated platelet activation only by blood contact with large volumes of polymeric material whereas both visual and computer-assisted evaluation of platelet spreading ability revealed a marked shift in the different classes of platelets: platelet activation results in a decrease of large structural elements and an increase of elements with spider threads. (pre contact (n=1000): 27:~-6 large forms of platelets, 700~-39 small forms and 275:l-41 spider forms; post contact (n=1000): 6-+-5 large forms, 510a:56 small forms and 484±58 platelets with spider threads). in some series there were significant differences between visual and computer-aided evaluation in the detection of small and spider forms. however, the relative increase of these nonspread spider forms could be stated with beth methods (wilcoxon test). we therefore conclude, that platelet morphometry with both methods is a sensitive and reliable ex vivo method to evaluate platelet interactions with artificial surfaces and can also be used lateron in phase-4-studies in patients. however, the ibas-system requires further maprovement in hard-and so,ware to reduce the high expenditure of this method. despite for the most part standardised methods such as hypothermia, cardioplegia the perioperative myocardial infartion rate is still high at approx. 6%. in cardiovascular surgery it is well known that various cardioplegic solutions are employed for myocardial protection during the ischemic phase. in order to evaluate the possible influence of these solutions we selected two of the most commonly used cardioplegic solutions for investigation in a randomised double-blind study: htk (group 1) and st. thomas (group 2). after randomisation each group consisted of 20 patients who had to undergo aortocoronary bypass surgery. aim of the investigation was to establish possible varying cellular changes during the reperfusion phase or in the early operative phase in order to be better able to apply reinforcing clinical measures. in the context of this study the classical enzyme-diagnostical methods ck,ck-mb and ldh as most useful, however not as convincing. still, we have in the meanwhile been able to show that the cardiac muscle troponin t proves a particularly sensitive parameter regards differentiated ischemic damage to the myocardium. ~his we were able to conflrm in extensive preliminary trials. cardiac troponin t was registered with a one-step lmmunoassay using two highly specific monoclonal antibodies directly via two different epitopes of cardiac troponin t. simultaneously the corresponding pre-and postoperative ecg was registered. further, within this context we investigated parameters that indicate cellular damage, such as platelet factor 4 (pf4), t-pa, interleukin-6 and pmn-elastase. in the reperfusion phase in group 2 there is a significant rise in tmponin t while in group 1 these values remain practically unchanged up to the 1st. postoperative day. of special importance is interleucin 6 since according to most recent studies the release of this substance leads to platelet activation via the arachidonic acid metabolism. this pathway must, further, be regarded within the context of free radical formation. on the 1st. postoperative day the 11 6 values in group 2 are significantly higher. the effects of membrane damage is also observed via pf4 and the pmn-elastase to be different in both groups. on the basis of this study we arrive at the conclusion that the htkcardioplegia is essentially less damaging than that of the st. thomas solution. (2) r. hetzer (2) (1) department of hematology and oncology, vimhow klinikum, humboldt university, berlin, germany (2) we investigated the influence of two different vad systems on these hemostatic changes. vads were implanted in 18 patients [11 bi-vad (berlin heart), 7 left vad (novacor n 100)] with end-stage heart disease who were awaiting heart transplantation. the following hemostatic parameters were measured during the first 51 days of bddging or until heart transplantation: thrombin-antithrembin iii (tat) complexes, prekallikrein, factor (f) xll, plasminogen, or2 -antiplasmin, and i?,thremboglobulin. results: during the first week of bridging, significantly higher tat levels were observed in novacor patients compared to berlin heart patients. prekallikrein activity levels were significantly lower in the berlin heart patients in the early bridging period. all other parameters were comparable in both groups throughout the entire observation period. differences in hemostatic parameters became apparent only in the early bridging period with more enhanced pmthrombin activation in the novacor group and more prominent contact activation in the berlin heart group. avoidance of the transmission of viral infections and saving in the use of blood products encouraged the use of apparatwe intraoperative autetransfusion techniques. patients and methods: arer randomization apparative intraoperative autotransfusion was performed in 5x7 patients during elective hip surgery using i-iaemonetics cell saver ill, haemonetics cell saver v, electromedics elmd, haemolite 3 and fresenius continuous autotransfnsion system (cats). at defined tmaes we detenmned a lab panel (clinical chemistry, lipids, proteolytic capacity, hemolysis, coagulation panel) at 9 determination points in the reservoir, the retransfused blood and in the patient. results: no significant differences concerning proteolytic capacity, prothrombin time, platelets, lipids, electrolytes. increased hemolysis (p<0.01) in the hcs iii group vs. the other groups (lo rain. after application of the retransfnsed blood). low heparin concentrations of retransfused blood in the hcs iii group( 0.32+-0.3 u/ml) vs. high concentrations in the cats group (0.47 +-0.3;p--0.01). parameters of thrombin generation were elevated in the hcs iii group vs. the other groups (p=0.02). conclusions: the use of 5 different apparative autotransfnsion systems dunng elective hip surgery results in dysturbances of hemocompatibility. the activation of the coagulation system during the collection and filtering is partly influenced by the elimination kinetics and the dose regime of heparin. however intraoperative autotransfusion must be roan~ged very carefully and possibly adverse effects of perioperadve heparin peak levels have to be considered. little information is available on the management of patients with factor viii deficiency who require cardiac surgery. we report the case of a 54 year old man with factor viii deficiency and combined severe aortic stenosis and incompetence and mitral incompetence who underwent a double valve replacement at our institution. he had a history of several bleeding episodes following minor surgery. previous factor viii levels were between 8 and 26%. using standard cardiopulmonary bypass, a double valve replacement with a 23 and 29 mm bileaflet prosthesis in aortic and mitral position, respectively, was performed. a high dose aprotinin regime was used (5.5 x 10 a iu). three doses of factor viii concentrate were given in the perioperative period, totalung 7000 1u until the 1st postoperative day. repeated measurements of the factor viii level were performed. the postoperative chest tube drainage was 350 rot. until the 4th postoperative day an additional dose of 3000 iu of factor viii was given to maintain a level of at least 30%. the obligatory anticoagulation was achieved initially with heparin i.v. in therapeutic dosage. due to a persistent 3rd degree av block a permanent pacemaker was inserted with additional 2000 iu of factor viii. on the 17th postoperative day warfarin was commenced aiming for an inr of 3.0 -3.5. the patient was discharged home therearer. he was trained to monitor his inr with a coagu chek device. no bleeding episode occurred during the first 3 months follow up. open heart surgery can be performed safely in patients with factor viii deficiency with the use of factor viii concentrates and monitoring of factor viii levels. coating of biomaterials was developed using synthetic polymers with incorporated anticoagulants. stents were coated with a thin layer consisting of a polylactide polymer containing peg-hirudin and a stable prostacyclin analogue. these materials were tested with a ,,human shunt model" using nonant/coagulated blood of healthy volunteers. within minutes uncoated stents were covered by fibrin and aggregated platelets, which could be seen macroscopically and by scanning electron microscopy; coated stents were free from coaguiation plugs. this observations were supported by analysis of coagulatiuon activation markers. unlike coated stents, uncoated stents revealed high levels (>detection limit) of tat complexes and prothrombin fragments (f1-2). in a series of experiments stents were tested in sheep. in 16 sheep stents (coated/uncoated patmaz-schatz stents) were ptaced by conventional techniques in the left anterior descending artery. anticoagulant therapy consisted of a heparin bolus and intravenously given aspirin before stent implantation. no ant/coagulation was given thereafter. existing data show hyperplasia in the area of uncoated stents which was reduced around coated stents (this study will be finished in january 1996). this coating technique with incorporated anticoagulants reduces thrombogenicity during the early and late phase of biomaterial implantation. studies concerning catheters, vascular prosthesis and oxygenators are in progress. the mechanical circulatory support (mcs) is a therapy for patients (pts) with endstage cardiac insufficiency. during mcs thrombeembolic events, due to the surface thrombogenicity of the implanted device, are feared complications. activated blood platelcts play a major role in this context. therefore, patient's platelet morphology was investigated. during the period of mcs, using the novacor left ventricular assist system n100, blood samples of 8 pts were observed by means of scanning electron microscopy (sem). blood was collected preoperatively and after implantation daily during the first week as well as weekly for the first 3 months. samples were drawn via an 18gauge cannula into caeodylic-acid buffered glutaraldehyde and platelets were prepared for morphological investigations. platelet alterations were classified as non activated, activated and aggregated, based on "shape change" morphology. additionally, the common blood coagulation parameters were evaluated. preoperatively, 15.0 + 4.6 % of activated platelets were found. within the first postoperative week, the mean level of activated platelets raised to 32.8 + 8.0 % (p<0.05). comparing short-(<30 days) vs. long-term (>30 days) mcs, a significant difference of activated ptatelets (overall mean values) could be seen (24.3 +_ 3.3 % vs. 34.8 _+ 3.4 %, p=0.004). during mcs a correlation between hemolysis and platelet aggregates, as well as the values of activated dotting time and activated platelets were observed. also, specific platelet deformations and damages appeared during mcs, which could not be found preoperatively. all pts with mcs showed alterations of their platelet morphology induced by the activation of the implanted synthetic material. with regard to the postoperative antithrombotic therapy, these observations should be taken into consideration. during extracorporeal circulation (ecc) the blood and its compenents are exposed to artificial surfaces and inflammatory respenses are activated, especially the complement, coagulation, fibrinolytic and kallikrein systems. furthermore leukocyte activation occurs and platelet function is impaired. these humoral and cellular systemic responses are known as the "pustperfusion syndrome" with clinical symptomes like lenkocytosis, increased capillary perraeability, accumulation of interstitial fluid and organ dysfunction. the impertance and even perhaps the existence of the damaging effects of cpb have been widely debated in the literature over the past 30 years. many efforts have been made to reduce traumatizing factors, e.g. the use of membrane instead of bubble oxygenators. recently, heparin-coated equipmen~ and tubings have been proposed to avoid excessive contact activation during cpb, the here presented study was designed to assess changes in coagulation and flbrinolytie activity in 20 patients undergoing cpb. in this regard we investigated coagulation parameters like fibrinogen, antithrombin, pmthrombin-fragments fl+2, thrombin-anthhmmhin complex, tissue-factor, fibrin-monomeres and parameters of the fibrinolytic system like tissue-plasminogen-activator, plasminantiplasmin-complex, d-dimers and plasminogen-activator inhibitor before, during and after cpb. the activation of the complement cascade was followed by measuring the concentration of c5a, c4 and c3c. the results demonstrate distinct alterations in above mentioned parameters. in spite of a high dose hepariulzation (act>450s) combined with an antifibrinolytic tw, atment an activation of the coagulation system was observed immediately after the onset of cpb followed by an activation of the fibrinolytic system. therefore further efforts should be done to develop new anticoagulatory regiments and improve the biocompatibility of materials used for cpb. during cardiopulmonary bypass blood is exposed to nonphysiologic conditions. the contact with artificial surfaces and mechanical stress result in a periopemtive response which includes activation of the complement, coagulation, fibrinolytic and kallikrein system, activation of nentrophils with degranulation and pmtease enzyme release, oxygen radical production and the synthesis of various proinflammatory cytokines. this so-called "pest-pump intlammatory response" has been linked to respiratory distress syndrome, renal failure and neurologic injmy. our goal was to investigate the time course of eytokine levels and the activation of leukozytes and platelets and to quantitate leucocyte subpepulatioas in 20 patients undergoing cpb. at different time points, pre, during and pest cpb, we determined the levels of interleukin (il) 113, il-2, il-4, il-6, il-8, il-10, tumor necrosis factor ¢z (tnf-a) and interferon "1' (ifn'--/) using elisa-techulques. lymphozyte subpepulations were characterized by flow cytometry and specific monoclonal antibodies against cd3 (pan t-cell marker), cd4 (surface antigen on t-helper cells), cd19 (surface antigen on b-cells), monocytes were determined by cd14 and platelets by cd41 (act. gpilb/llla) and cd42b (gp ib). single cell activation was analyzed using markers against cd25 (il-2 receptor), cd126 (il-6 receptor), hla-dr (mhc class ii), cd71 (transferrin receptor) and cd69 (activation inducer molecule), platelet activation was monitored with an antibody against cd62 (gmp-140). preliminary results revealed distinct increases in r,-6, il-8, and il-io following cpb whereas tnf-a and ifn--/levels were not significantly influenced. fttnhermore, activation of particular cell populations was observed. finally, our investigations should contribute to a better understanding of the complex humeral and cellular respenses induced by cpb and thus might help to develop new strategies to circumvent the negative impacts of cpb. optimal adjustment of anticoagulation in machine plasmapheresis is important for the quality of the prepared fresh frozen plasma (ffp) as well as for the safety of the donation. in the present study the suitability of prothrombin fragment ( ft+2 ) in the assessment of anticoagulation during plasmapheresis was investigated. matarlal and methods: 75 plasmapheresis procedures were performed on 25 donors (10 ~, 15o" ) using 3 different plasmapheresis machines (a 200, baxter; mcs 3p, haemonetics; pph 900, electromedics/medtronic). acid citrate dextrose formula a (acd-a) in a ratio to whole blood of 8 : 92 was used for anticoagulation. the concentration of fi+2 in the donor's blood was measured before and after plasmapheresis and in the prepared ffp. the actual acd-a volume used was also registered. results: there was a significant rise of the ft+2 -concentration in the donors blood after plasmapheresis with each of the three automatons: a 200:1.32 vs 1.14, p < 0.05; mcs 3p: 1.26 vs 0.98, p < 0.05; pph 900:1.20 vs 1.05, p < 0.05. the ffp prepared with each machine showed the following f~+2concentrations: 0.91± 0.18, 1.0:2 ± 0.17 and 0.93 ± 0.11 respectively. the difference between the groups was not significant. the elevation of the ft+2 -concentration in the donor's blood showed a negative correlation with the volume of the acd-a used. during 6 of the 75 procedures technical problems occurred (inadequate venous acces, occlusion of the citrate tube, reduced whole blood flow). after these procedures there was a marked elevation of f~+2 in the donors blood (2.74 ± 0.53), accompanied by an elevated f~+2 -concentration in the prepared ffp's. conclusion: these data show that ft+2 is a suitable parameter for the assessment of anticoagulation during plasmapheresis. several epidemiologic studies demonstrated that fibrinogen is an independent cardiovascular risk factor and should be considered for screening programs. prothrombin time derived fibrinogen (df) measurement combines the advantage of an established highly reproducible automated method with no additional reagents, except for calibration. several studies showed that the df values correspond well with the clanss method except in cases such as thrombolytic therapy in which the df results are higher. however, no results exist whether in patients with coronary heart disease with fibrinogen as a risk factor the df values are also comparable to the established clausss method. the aim of our study was to compare df values to clauss method results in cardiac patients, especially in patients before and after coronary bypass grafting (cab(]). measurements of df were performed on an acl 3000 (il) using the pt-fibrinogen-hs reagent. fibrinogen clanss method was done on the acl using fibrinogen c reagent (il) and on a kc4 (amelung) with fibrinogen a reagent (boehringer maanheim). for calibration we used the calibration plasma half volume (it.) with the fihrinogen concentration proposed by the manufacturer. plasma samples were obtained from 24 patients at admission before cabg and postoperatively up to 1 week, and from 23 healthy persons (staff). within assay imprecisious using normal and abnormal controls (il) were comparable with both methods showing cvs between 1.99 and 4.22 %. in normal healthy persons the medians of the df and the clanss method run on the acl were very similar (296 vs 302 rag/all), whereas kc4 values were about 10% lower (268 md/dl). in cabg patients at admission we found the same differences as in normals with the clanss method (acl: 363 vs kc4: 337rag/all), however the df values were siginficantly higher (median 418mg/dl). if we took a cutoff value of 320 mg/dl, as suggested by the results from the northwick park heart study, we would categorize into the high risk group 21 out of 24 patients using the df method, 20 with the clanss-acl method and 16 with the clanss-kc4 method, i.e. nearly 30% more patients were classified in the high risk group using the df method. postoperative samples showed the expected increases due to the acute phase response with the same magnitude of differences. because of its rapidity and reproducibility the df method is well suited for routine measurements, however, standardization remains an urgent task in order to avoid misinterpretation of results. for fibdnogen measurements in clinical laboratories, the two most widely used methods are the clotting time method according to clauss (cfib) and the sn called "derived" fibrinogen method (dfib) implemented in optical coagulometera with the fibrinogen concentration being derived flora the optical density of the fibrin clot in a standard prothrnmbin time (pt) assay. it is well known that under certain circumstances, e.g. in the presence of fibrin(ogen) degradation products (fdp), there is a discrepancy between the two methods with higher values for dfib than for cfib. yet the opposite discrepancy, i.e. fibrinogen values derived from the optical density of the clot grossly lower than values from dotting time assays, seems to be very rare and is poorly understood so far. the patient (male, 26 years) had ingested the esterase inhibitor parathion (e605) in an attempt o f suizide and was treated with high doses of atmpin. he had no clinical signs or history or family history of bleeding or thrombotic disorders. except for a very low pseudocholinesterase activity, all laboratory results were normal ineinding pt, afft, thrombin time, and factor xiii. pt and aptt did nnt differ between an optical coagulometer (electra 1000c, mla) and a mechanical one (kc..4, amelung). there was no evidence of disorders known to interfere with hemostasis like paraproteinemia or dyslipldemia. however, in all 7 blood samples received for dotting tests during a period of 7 days the macroscopic appearance of the fibrin clot was quite unusual (only slightly turbid/almost transparent) and there was a striking discrepancy between a very low or low dfib on the electra (pt reagent: thromboplastin is, dade) and a normal or high cfib (kc4; thrombin reagent, dade). on admission, values were 57 mgml (derived) vs. 275 mgldl (clauss). cfib rose to s42 mg]dl with dfib at 155 mg/dl in the last sample on day 7. ~ al! samples dfib was about 20 % (ls-23) of cf[b. when the patient's plasma was added m normal pooled plasma it caused, in a dose-dependent manner, values lower than predicted for dfib and values slightly higher than predicted for cfib. in the absence of data from additional (e.g. immunologic) methods the following principal possibilities (and combinations) have to be considered: 1) normal fibrinogen concentration and clot formation rate, but abnormal optical properties of the clot (cfib correct, dfib falsely tow); 2) normal optical properties of the clot, but accelerated clot formation and very low fibrinogen concentration (dfib correct, cfib falsely high). in either case, the molecular basis could be: a) a genetic or acquired molecular abnnrmality of fibrin/fibfinogen; b) an interfering substance. direct effects of the loxic agent parathion and/or the antidot drug atropin are not likely to be the cause since other patients, often with more severe parathion inmxicatian requiring higher doses of atmpin, showed normal optical density of the clot. we hope to perform a more in depth investigation of this abnormality in the future, including various methods, reagents, and instruments for fibrinogen measurement, a survey of the patient "s family, and studies of the molecular nature of the phenomenon. increased fibrinogen is known to be an independent predictor of subseqtmnt acut~ coronary syndromes. however. a multitude of methods for fibrinogen determination is available. there is a lack of standardisation among fibrinogen assays. in a family cohort study (patients'with combined hyperlipidaemia and f or hypemricaemia) fibrinogen was determined in plasma samples from 340 family members using a functional and an immunochemical assay. the fimctional assay according to clauss was performed on the analyser ca 5000 using the test fibrinogen a from boehringer. the immmmephelometric assay was performed on ~e behring nephelometer system using the reagent and standard from behring. a good similarity between both assays was obtained at low and high flbrinogen levels as well as in samples with increased c-reactive protein (crp). values obtained by both assays correlated similar with total cholesterol, ldl--cbelesterol and apolipeprntein b. the ratio functional fibrinagen / immlmochemial fibrinogen showed no dependence on cholesterol, t-pa, v wiuebrand factor and crp. release of two fibrinopeptides a from fibrinogen generates desaa-fibrin monomer, which rapidly aggregates, forming fibrin complexes. fibrin monomers can be detected in plasma samples after chemical desaggregation of fibrin complexes using thiocyanate by monoclonal antibody binding to the alpha-chain neo-n-termini generated by fibrinopeptide release. although postulated, an intermediate of fibrin formation, carrying one fibrinopeptide a and one fibrin alpha-chain neo-n-terminus has so far escaped analytical procedures. we have employed a monoctonal antibody specific for fibrin alpha-chain neo-n-terminus, mab 2b5, attached to magnetic microparticles, for isolation of fibrin-related material from plasma samples of patients with elevated soluble fibrin. the material was desorbed by sds-urea buffer and subjected to sds-page and immunoblotting. immunostaining with panspecific anti-fibrinogen and anti-fdp-e antisera showed a range of bands corresponding to fibrin monomers, and fibrin derivatives containing the fibrin e-domain. lmmunostaining with monoclonal anti-fibrinopeptide a antibody resulted in a doublet band corresponding in size to fibrin monomer. similar results were obtained with polyclonal antisera against fibrinopeptide a. for a more quantitative approach, desa-fibrin monomer was detected by an elisa procedure using mab 2b5 as capture and monoclonal anti-fibrinopeptide a antibody as tag. a sample with extremely high level of desaa-fibrin monomer, determined by elisa (enzymun®-test fm) was used for calibration, since reference material is not available. a correlation of r=o.g4 was found between desaa-fibrin monomer and relative desa-fibrin monomer levels. detection of desa-fibrin monomer required sample pretreatment with thiocyanate for desaggregadon of fibrin complexes. from these preliminary data it appears that desa-fibrin monomer accounts for a fairly constant proportion of soluble fibrin and is a polymerizing species. fibrinogen has been shown to be a major cardiovascular risk factor. especially for epidemiological studies, exact quantitation of fibrinogen in clinical plasma samples is of great imporance. fibrinogen levels are generally measured by clotting assay according to clauss, or by determination of derived fibrinogen values upon photometric measurement of prothrombin time (derfbg). the clotting assay has been shown to be influenced by high levels of soluble fibrin derivatives. the pt-derived fibrinogen levels appear rather convenient in clinical routine, since no additional reagents are needed. we have compared the clauss assay and derfbg with a turbidimetric fibrinogen assay using snake venom protease for fibrinopeptide release, performed in photometric autoanalyzers. d-direct antigen was measured in parallel using tinaqaant d-dimer lpia. results were correlated with total fibrinopeptide a release by thrombin, measured by elisa. a total of 484 samples were included, of which 29 samples (6 %) were recorded as above measurung range by derfbg. these samples encompassed a range of 5.90-10.40 g/l and 5.21-12.37 g/l in clauss, and turbidimetric assay, respectively. the range of values measured by derfbg assay was 0.72-9.14 g/i, corresponding to 0.26-11.00 gll and 0.24-10.48 g/1 in the clauss and turbidimetric assay, respectively. the correlation of derfbg with the clauss assay was re0.91, correlation with turbidimetric assay was r=0.92 for the values actually detected. the correlation between clauss and turbidimetric assay was r=0.93 for all values. there was no dependency of test results or inter-test variation upon d-direct. correlation graphs displayed a decreased test response of clauss assay in the high concentration range, resulting in an underestimation of fibrinogen concentration. the derfbg assay, in contrast, showed normal range values in samples from patients with fibrinotytic treatment and low fibrinogen levels in the other assays. correlation with fibrinopeptide a release was r=0.88 for clauss assay, r=0.89 for turbidimetric assay, and r=0.82 for derfbg. for clinical routine, derfbg appears to be applicable for all samples between 1.00 and 5.00 g/l with exclusion of samples from patients with fibrinolytic treatment or endogeneous hyperfibrinolysis. other samples may be analyzed by clotting assay or turbidimetric assay, although the latter appears to be more suited for measurement of high range samples. for inhibition of pk is 0.067 pmol/l the antifibrinolytic activity of the inhibitors was determined by measuring the lysis of radiolabelled human plasma clots• the compounds which inhibit plasmin and pk influence remarkably the streptokinase-induced clot lysis but not lysis induced by uk and tpa. surprisingly, inhibitors of uk and tpa do not influence clot lysis induced by uk or tpa. the structure-activity relationships for inhibition of ptasmin, uk, tpa and pk could help in the design of more potent inhibitors of fibrinotytic enzymes. uk inhibitors are of interest for the development of anti-invasiveness drugs, while plasmin/pk inhibitors could be prototypes of a "synthetic aprotinin". in the ecat angina pectoris study t-pa antigen was an indepcndem risk factor of subsequent acute coronary syndromes. pat indicates the risk bat depends on other known risk factors. it should be tested in 183 members of a family cohort study (patients with combined hyperlipidaemia and / or hyperuricaemia), if the active pal antigen or the whole pai antigen showed a stronger relation to t-pa and metabolic variables. the active pall antigen was determined using elisa actibind pat-1 (technoclone / lmmuno) , the whole pai-i antigen was measured using the f_lisa pat-1 (technoclone i immuno). t-pa activity was determined with the coaset t-pa from chromogenix, the tintelize tpa from biopool was the used test for determination of t-pa antigen. the active pat antigen showed a stronger correlation to t-pa activity and t-pa antigen than the whole pal antigen. circulating t-pa activity was influenced predominantly by the active pal antigen. both pat antigens were correlated in similar manner with metabolic variables, lipoproteins and b/vii. table: correlations of active and whole pal antigen (** p < 0,001) active pal antigen whole pal antigen active pat antigen 1,000 0,851 ** whole pal antigen 0,851 ** 1,000 t-pa activity -0,594 ** -0,492 ** bpa antigen 0,604 ** 0,497 ** body mass index 0,502 ** 0,462 ** triglycerides 0,452 ** 0,441 ** total cholesterol 0,252 ** 0,255 ** ldl-cholesterol 0,263 ** 0,264 ** hdl-cholesterol -0,357 ** -0,355 ** apolipoprotein b 0,428 ** 0,402 ** apolipoprotein a i -0,233 ** -0,211 ** the lower relationship of the whole pat antigen to t-pa is obviously caused by patient samples with high levels of whole pat antigen in contrast to normal values of active pat as well'as of t-pa. possibly, a high ratio of whole pai antigen / active pat antigen is caused by a raise of latent pal the main form of pat in the platelets. the clinical importance of an increased ratio whole pal antigen / active pal antigen remains under investigation. the cyclic antibiotics-polypeptides bacifracin a, bacilliquin from boci/lu~, licheniformis and gramicidin s from bocil/us brevis, var. (3. b., were used for investigation. we studied their influence on the fibrinoly~c and coagulation activity in vitro• me~hods. to solution of human plasmin (thrombin). containing 0.2 mg of protein (1 nih unff)/ml, the analyses' solution of antibiotics (0.1-8,0 mg) was added. then we defined the tlbrinolytlc activity of the mixes using azofibrin lysis, and fhrombin activity was determined according to the speed of fibrin clots formation from fibrinogen solution. results. in following table are submifled the results received in our laboratory {we also offer results of antibiotics influence on urokinase activity): ki, mm ki --the constant of inhibition. n. d. ~ in studied lirnils the inhibitor's activity was not observed. ---the inhibitor's activity was not define. i. --the inhibitor% activity was observed but ki not determined. +, +% +++ --effect of inhibffion (in rela*iive indexes). conc/us/on.~ the results received by us testify to the necessity of cautious approach to the use of antibiofics-polypeptides for various sorts of therapy in view of their possible influence on fibrinolytic and coagulation actlvlfy, of the organism. these results were used for preparation in our laboratory of biospeciflc sorbents containing c-ramicidin a, bacil}iquirt and gramicidin s.as ligands, they can reversibly bind thrombin, plasmin {plosminogen) and urokinase directly from crude exkacts. the enzymes are selectively eluted without substantial losses of specific activity in e yield of 60-90%. there is a great body of rather contradictory informations dealing with fibrinolysis in liver.. cirrhosis, which can be accelerated, normal or reduced, depending on the type of cirrhosis and investigation techniques (clot-lysis, fibrinolytic component measurements). our previous finding was, that in vitro plasma-clot lysis, induced by exogeneously added tpa or streptokinase proved to be reduced, and this had a good correlation with severity of the disease and the elevation of plasmatic yon willebrand factor levels. in vitro clo~/-[ lysis tests, induced by tpa were performed in 41 patients with alcoholic liver cirrhosis, utilising a microplate light-scattering assessment method. the tests were repeated using the same plasma samples in each patients with a microplate which was covered by cultured endothelial-cell monolayer (umbilical vein, huvec}. clot lysis speed proved to be 1.5-2 times slower with huvec milieu in the control group, while in the cirrhotic patients this inhibition was stronger and resulted in 5-fold reduction of lysis speed. our results suggest, that cirrhotic plasma is able to accelerate the release of fibrinolytic inhibitors from cultured endothelial cells, which phenomenon may also contribute to the complex alterations of in vivo fibrinolysis in cirrhotic patients. deep vein thrombosis (dvt) is a systemic disease with prolonged clinical manifectation. anticoagulation therapy in dvt is not completely effective. thrombolytic therapy may give rise to a systemic lytic state, the fibrinospesific agents (scu-pa and t-pa) have short half-lives in the circulation. we investigated the potency of the acylated plasminogen streptokinase activator complex (gbpg-sk) to deep vein clot dissolution as compared to well known sk and apsac both in v~tro and in vivo in the model of venous thrombosis in artherio-venous shunt in rats. it was shown in in vitro study that fibrinolytic activity of plasminogen activators mainly depends on their stability in plasma. stability studies carried out by incubating sk and pg-sk activator complexes in plasma with euglobulin precipitation . total fibrinolytic activity was measured by the fibrin plate method. gbpg-sk possessed the greatest stability in human plasma than apsac or sk because of its prolonged inactivation period (the deacylation half-life for gbpg-sk was 230 :e 21 rain in contrast with 73 -~ 6 min for apsac). the stability degree of two acylated thrombolytics (gbpg-sk and apsac) was in order to inverse proportion of their first order rate deacylation constants (2.9 • 10 -4 and 6.0 • 10-s sec-1 respectively). the fibrinolytic potency of sk, apsac and gbpg-sk was measured by 1251-labeled fibrin clot lysis in plasma and in vivo by lysis of the preliminary formed 1251-labeled fibrin clot inserted into the jugular vein. fibrinolytjc activity of acylated plasminogen activators gradually increased in time. under sk administration, the clot lysis came to the end by 2 hours while apsac and gbpg-sk haven't lost their activity for 5 -6 hours. gbpg-sk possessed significantly more prolonged fibrinolytic activity than apsac, the acyl-enzymes did not significantly influence on plasminogen,,.~2-anfiplasmin and fibrinogen levels in plasma according to their activity specific to fibrin-bound plasminogen. in opposite, sk produced a significant depletion of plasminogen, ~-2antiplasmin and fibdnogen levels in plasma. it seems, on the basis on in vitro and an animal experimentation, than apsac with its moderately fast deacylation rate is more suitable for rapid thrombolytic effect, but gbpg-sk with its slow deacylation rate is suitable for deep vein thrombosis, when the rapid thrombolysis is less critical. it's well known that the complete lysis of thrombi usually isn't observed at the thrombolytic therapy. at present study we have attempted to quantify the possible mechanism of fibrinolysis inhibition during the thrombolysis. 125i-labelled partially cross-linked fibrin clots of different volume (0.1-0.35 ml) were immersed in tris-hcl buffer (3 ml) containing plasmin (5-100 nm) at 37°c. the lysis rate was detected by counting of soluble fibrin degradation products (fdp). at all the eases lysis slowed down and stopped in 3 hs though clots dissolved up to only 60-85%. no irreversible inlaibition of plasmin caused by denaturation occur as was judged by the measurement of fibrinolytic activity at the diluted samples. however the increase of fdp concentration in surrounding buffer led to the reversible inhibition of fibrinolytic activity of plasmin up to 5% of baseline. the sds-page analysis under non-reduced conditions shown the acoumulation of high-molecular weight fdp at the surrounding buffer. the inhibition phenomenon could be connected with the specific binding of plasrnin with soluble fdp having exposed lysine residues and the subsequent removal of enzyme from fibrin surface. unexpectedly since the heterogeneous character of occurred reactions tile change of the clots surface area during lysis didn't affect the fibrinolysis kinetics in all the concentration intervals. to estimate the kinetic parameters the kinetic curves were linear in the coordinates [p1/t (l/t*ln(isl°{(lslo.lpi)). the obtained parameters were following: keat=l.36 min-l,km=l.33 ixm,kp=0.12 ~tm. the clinical trials have shown that fdp concentrations at the thrombolytic therapy of deep venous thrombosis and acute myocardial infarction usually was approximately in the range 0.05-0.2 ~tm. therefore the described phenomenon of fibrinolysis inhibition by formed fdp may take place during thrombolytic therapy. al. calatzis, an. calatzis, +m. klmg, +l. mielke, +r. hipp, a. stemberger institute for experimental surgery and +institute of anesthesiology technische universit~.t monchen thrombelastography (teg) is an established method for the detection of fibrinolysis. fibfinolysis is usually determined when the teg amplitude decreases by more than 15% atter the maximum amplitude is reached. this takes a considerable amount of time (more than 30 minutes). our approach bases on the understanding of fibrinolysis as a process which runs in paraue[ to coagulation and is not exclusively subsidiary to it. the effect of fibrinolysis on the growing clot in the teg is shown by the comparison of two parallely performed teg measurements: exteg: teg measurement with standardised activation of the extrinsic system. apteg: exteg with in-vitro-fibrinolysis inhibition via aprotinin. exteg-reagent (ex): 1:2 dilution of innovin (recombinant thromboplastin reagent, dade) with aqua dest. apteg-reagent (ap): 5 parts innovin, 2 parts trasy[ol (aprotinin, bayer, i0.000 kie/ml), 3 parts aqua dest. test procedure: l0 p1 ex or ap + 300 ~l citrated blood (cb) + 50 lal cacl2-solution 0,15 m. the only difference of the two reagents is the addition of 20 kie aprotinin in the apteg, leading to an in-vitro fibrinolysis inhibition. the usage of disposable pins and cups (haemoscope, illinois, usa/e.m.s., vienna) is recommended for ensuring standardised conditions for both measurements. results and discussion: when there is a better clot formation in the apteg (corresponding to a lower so-cafled k-value) than in the exteg, fibdnolysis can be suspected. this technique requires only commercially available reagents and is easy to perform on conventional teg systems. due to the standardised coagulation activation with a thromboplastin reagent, fibrinolysis can be detected also when inhibitors like heparin are present in the circulation. according to our experience using this technique during liver transplantation, clinical relevant fibrinolysis can be detected as described in less than l0 minutes. many thromboembolic (massive pulmonary embolism, proximal deepvein thrombosis, etc.) and coronary diseases (infarction, acute phase, etc.) require fibrinolytic therapy to early recanalizafion. the application of the well-known or new thrombolytic agents needs the use of specific, simple and reproducible methods for the determination of fibdnolyfic activity. we suggest new methods for measuring the blood plasma concentrations of plasmin, plasminogen, antiplasmins, and urine urokinase activity. these methods involve the employment of chromogenic substrafe azofibrin (human fibrin, covalently labeled with p-diazobenzenesulfonic acid). method~. 0.2 ml of studied solution was added to 0,8 ml of azofibrin suspension in certain buffer (5-10 mg/ml) and the mixture incubated at 37 oc for 10-60 rain. after the end of incubation the mixture was filtered, the volume of solution brought up to 4 ml by 0.02 m naoh and the optical density was determined at 440 nm. resuffs. azofibrin can be used for quantitative determination of proteinases activity in search of new fibrinolytic means. for comparison the results of our studies fibrinolytic activity of some proteinases with the use of azofibrin are presented: activity. with an increase of pal and ldl-and a decrease of hdl-cholesterol concentrations k is concluded that the increased cardiovascular risk in diabetes meilitus was partly caused by a down regulation of the fibrinolytic system, increase of erythrocyte aggregation and plasma viscosity. also disturbances of lipid metabolism an abnormal whr seems to be of an additional atherogenous factor in dm. plasma concentrations of thrombin-anfithrombin-iii (tat), alpha-2antiplasmin-plasmin (app) complexes and ddimer were investigated in 50 patients treated with thrombolytic therapy for acute myocardial infarction (ami) either with streptokinase (n=24), urokinase (n=16) or recombinant t-pa (rt-pa, n=10). all patients received an intravenous heparin bolus of 5,000 iu on admission, which was followed at once by an infusion of 1,000 iu/hr for the next three days titrated to maintain the partial thromboplastin time at twice control value. tat, pap and ddimer were measured by enzyme immunoassay on admission, 1, 2, 4, 6, 8, 12, 24 hours and on day 3 and 7 after admission. groups did not differ significantly in regard to age, sex, delay and infarct location. on admission, no marker differed significantly between groups. thereafter, tat levels increased significantly exclusively in rtpa treated group. from 2 to 6 hours after admission, tat were significantly higher in rtpa treated patients than in streptokinase and urokinase treated group (p<0.02). however, during continous heparin infusion, which was started immediately after stop of thrombolytic therapy, in each group tat concentrations decreased below admission values. app were significantly higher only 1 hour after admission in the rt-pa group (p=0.03). ddimer did not differ signifieanfly between groups. our results demonstrate, that rtpa induces a hypercoagulable state, which may contribute to reocclusion after successful reopening of the infarctrelated coronary artery. the significant tat decrease during continous heparin infusion supports the concomitant use of thrombin inhibitors as adjunctive therapy with thrombolytlc treatment for ami. thus, in acute myocardial infarction patients, thrombin generation is markedly influenced by the thrombolytic agent used and concomitant heparin therapy. endothelium derived relaxing factor-no (edrf-no) plays a major role in regulation of vascular tonicity and also exerts platelet inhibitory action~ however, due to the chemical nature of edrf-no few is known about its production and activity as a general index or marker of vascular function in human diseases. one way to achieve this can be measurement of nitrate/nitrite excretion in the urine, which seems to reflect vascular edrf-no production. in this report a self-developed elisa method is described, which was used for this perpose. nitrate/nitrite urinary exretion proved to significantly decreased in insulin dependent and in non-insulin dependent diabetes mellitus as well after a comparison of the excretion values to other markers of angiopathy (yon willebrand factod soluble thrombomodulin, beta -thromboglobulin) it seems to be acceptable, that urinary nitrate/nitrite excretion can be a useful indicate of diabetic vascular disorders. two major concerns still accompany the application of prothrombin complex concentrates (pcc). viral safety has to be guaranteed and therefore several measures for virus inactivation or elimination are taken during the manufacturing process. the inherent risk of thrombo-embolic side effects has to be considered. to minimize these risks and to achieve good clinical efficiency the quality criteria for pcc's are under pending discussion. it is generally accepted that a modem pcc-preparation should contain all of the four coagulation factors in a well balanced proportion and that it should also contain protein c and protein s. additionally, the concentration of activated coagulation factors should be kept at a minimum. a present pcc-produedon process mainly consists of a qae-sephadex extraction of cryopeer plasma followed by a solvent/detergent virus inactivation step. further purification is achieved by subsequent chromatography on deae-sephamse. the aim of this study was to improve product quality by avoiding f viiactivation without implementing major changes to the production process. at the same time, a second virus eliminating step was added to the production process. it could be shown that speeding up the chromatographical process by switching the deae-sepharose-chromatography from a classical axial column to a radial chromatography resulted in a significant reduction of f viia-genemtion. mainly the reduction of contact time, resulting from the highest possible flow rates, leads to the wanted effect. the relation between f vii/f viia was 10 : 1 or more. in order to investigate the feasibility of virus filtration the eluate of the deae-sepharose column was filtered through a virus removing ultipor vffilter. the analysis of the solution before and after fillration showed that the filtration had no influence on coagulation factors activity, protein content, proteolytic activity etc. preliminary studies showed significant virus reduction values. in the past few years the problem of expediency of the treatment aimed at developing immunological tolerance in hemophil;a patients by way of complete removal of inhibitor with high doses of factor viii has been discussed in literature. we observed 121 patients with hemophilia. inhibitors to factor viii:c were revealed in 32.7 % of patients with hemophilia a and fo factor ix --in 1.6 % of patients with hemophilia b. the level of an inhibitor was not higher than 87 befhesda u/ml, that is those patients were not regarded as "high responders". a high incidence of inhibifors in young patients [from 7 to 26 years of age, 51.9 %) compared with older patients (from 27 to 40 years of age, 11.2 %) testifies to the probability of inhibitors development during treatment with modern concentrated preparation of factor viii, ix. inhibitor development in patients (40.5 %] in the course of antihemophilic concentrates transfusions is an evidence of alloimmunization of patients with proteins. the investigations show that in the course of transfusion therapy patients develop secondary immunodeficiency due to chronic antigenic stimulation of immune system with high doses of allogenic proteins. against the background of immunodeficiency patients with hemophilia develop complications of immune character: infections complications --53.9 %, aufoimmune processes --44.9 %, secondary tumours --1.2 %. plasmapheresis is the most rational method of removing inhibitor in patients with low level of inhibitor ("low responders", < 10 bu/ ml) and in patients with mean response. thus it should be noted that the treatment of patients aimed at developing immunological tolerance is not only expensive and economically unprofitable but also not indifferent fo the organism. in a recent multicenter study 73 previously untreated patientens (pups) with severe hemophilia a were treated with a recombinant factor viii concentrate (rfviii, recombinate©). during fviii treatment 21 (29%) developed inhibitors, 6 high titer (>5 bethesda units (bu)/ml), 4 low titer (<5 bu/ml) and 11 transient inhibitors. plasma samples from before treatment and during treatment but before inhibitor occurrence were available in 12 inhibitor patients. these plasma samples were analyzed by a highly sensitive immunoprecipitation (ip) assay for the presence of anti-tviii antibodies. in 9 (66%) a significant increase of anti-fv]]i antibodies was seen indicating the development of a clinical relevant inhibitor titer. this immune response occurred after 2 to 17 (median 5) exposure days (ed). in the same period only 3 out of 15 inhibitor patients showed a decreased in vivo recovery. in 16 pups who developed no inhibitors plasma samples from the entire treatment period were available. an immune response to rfviii treatment was seen in 7 pups after 2 to 43 ed (median 24 ed). the immune response was later and less pronounced in comparison to inhibitor pups before inhibitor occurrence. with the ip method the detection of an early immune response is possible which might be predictive for a later inhibitor development. the inclusion of the lip method should be considered for future multicenter pup studies. in the past anaphylactie reactions to plasma and plasma components have been a common complication of replacement therapy in patients with hemophilia a and b. we report on 3 severe bleeding episodes in 2 patients with hemophilia a and b, respectively. both patients had a history of life threatening anaphylactic reactions after exposure to different plasma derived clotting factor concentrations including intermediate purity factor viii-and factor ix-concentrate, respectively. high purity factor concentrates were tolerated well without any allergic side effects. a 67 years old patient with a moderate form of hemophilia a (f viii 4 %) had a history of severe immediate reactions with skin manifestations and bronchospasm after exposure to fresh frozen plasma, ctyoprecipitate and 3 different plasma derived factor viii-concentrates of intermediate purity. in all episodes pretreatment with corticosteroids and antihistamines was unsuccessfull in avoiding severe bronchospasm. replacement therapy with two different recombinant factor viii concentrates was tolerated well without any side effects. a 12 years old haemophiita b patient developed hypersensitivity reactions to prophylactic factor ix substitution, which could be overcome by using a factor ix .concentrate with improved purity. a recent recurrence of hypersensitmty under this treatment was finally overcome by the use of highly purified (monoclonal antibodies) factor ix concentrate. we conclude from these findings that high purity of factor concentrates, possibly due to the absence of soluble hla-antigens, are advantageous in patients disposed to allergic reactions. introduction: antibody formation against factor (f) viii remains one of the most severe complications of repeatedly transfused patients with haemophilia a. as reported previously in our study about the incidence of fviii inhibitors, we have observed a high incidence of fviii inhibitors among our haemophilia a patients. it is still not clear why certain haemophiliacs develop antibodies and others do not. a number of previous studies suggest that there is a genetic predisposition for the fviii inhibitor development. thus, the purpose of our study was to examine, if there is a correlation between fviii antibody-formation and genetically determined histoeompatibility antigen (hla) patterns in our haemophiliacs. patients and methods: hla-class i (a, b, c) and hla-class ii (dr, dq) typing was carried out for 51 respectively 44 multi-transfused paediatric haemophilia a patients (fviii:c activity < 5%), including 22 who had developed an antibody to fviii: 19 were high responders (> 5 bu), 3 were low responders (< 5 bu). hla-typing has been performed by a standurcl two-stage microlymphoc~.ftotoxicity procedure (drk frankfurt) using antisera with defiend hla-specifity (biotest diagnostica). results: we found an under-representation of hla-a2 in fviii inhibitor patients when compared with the subgroup without inhibitor. in regard to the hla-b and hla-c antigen frequencies there are no apparent differences between the groups. among the class ii antigens there were higher frequencies of dr1, drw52 and dqwl in the non-inhibitor group. however, the reduction in hla-a1, hla-cw5, hla-dqw3 respectively hla-dr4 frequency for inhibitor patients as reported previously could not be confirmed in our study. conclusion: so far it remains unclear if there is a significant association of a certain hla allels with the development of fviii antibodies. recombinant factor sq (r-viii sq, pharmacia) is a b-domain-deleted recombinant factor viii. it is formulated without albumin (hsa). the product has been shown to have in vitro and in vivo biochemical characteristics similar to a plasma derived full-length protein (p-viii). the international clinical trial programme was initiated in march 1993. pharmacokinetic studies have shown that the b-deleted r-viii sq should be given according to the same dosage principles as a full length p-viii. at present, the product is being tested in previously treated patients (ptps) and untreated patients (pups) with severe haemophilia a (viii:c < 2 %), both during long-term treatment (on demand therapy or prophylaxis) as well as during surgery. the long-term study in previously treated patients in germany was started in january 1994. thirteen patients have been included in 8 centers. all patients are still on treatment with r-viii sq, most of them receiving prophylactic treatment. global treatment efficacy has in general been considered excellent or good. no serious clinical adverse events related to the study product have been reported, nor have any inhibiting antibodies to factor viii or antibodies to mouse-lgg or cho-cell components developed in the patients. further results such as data on efficacy, half-life, recovery and safety will be presented in detail at the meeting. nowadays it is not sufficient to regard hemophilia only as hemorrhagic diafhesis of coagulation genesis, caused by deficiency or molecular anomalies of coagulation factor, without taking into account the immunity state. on examination of 125 patients (pts) (hemophilia a --110 pts, hemophilia b --11 pts, willebrandt's disease u 4 pfs) the development of immune complications was revealed in 34.4 %. chronic persistent hepatitis (3.2 %), chronic active hepatitis (3.2 %), herpes simplex (1.2 %), chlamidiosis (1.2 %), bacterial infection (7.2 %} were regarded as infectious complications. bacterial infections have a routine course due to preserved phagocytic function of neufrophils. and viral infections, whose ability to resistance is connected with t -cell link immunity, take on a chronic persistent course, mechanism of the development of autoimmune processes (autoimmune thrombocytopenic purpura --2.4 % of pts, immunocomplex disease --4.9 % of pts, the appearance of immune inhibifors --34.4 % of pts} is connected with the impairment of immunological surveillance over b -cells aufoimmune clones as a result of dysbalance in the system of t -lymphocyfes immunoregulatory subpopulations. lymphadenopathy and splenomegaly (4.9%) develop due fo benign proliferation of lymphoid tissue as a result of impairment of regulatory function of t -lymphocytes system, or they may be an evidence of virus infection. we observed one episode of acute leukemia. immune complications in hemophilia patients develop against the background of secondary immunodeficiency caused by chronic antigenic stimulation of patients' immune system with high doses of allogenic proteins, which plasma preparations contain. in immune complications hemophilia patients develop hemorrhages, whose pathogenesis is quite different from that caused by coagulation factor, so it should be taken into account in the course of treatment. control of hemophilia therapy classically was based on four parameters: life span expectancy of patients, orthopedic status (normal zero), pettersson score and social integration. oren, however, these parameters described an irreversible status with permanent damage particularly of the joints, especially when patients were grown-up. in order to establish risk-adapted therapy protocols to prevent hemophllic osteoarthropathies, quality control programs have to he set-up that allow for early adjustment of dosage and substitution frequency. here bleeding frequency is one the main parameters, being a clear hint for the possible development of a target joint. since 1988 we have established a computer database (haemopat) that contains data from all patients treated in our center. tables and graphs allow for early detection of increased bleeding tendency in a given joint, and accordingly for adjustment of therapy. the results of 8 years of measuring reasons of joint damage and not documenting the orthopathies as such will be demonstrated. parallelly a new program (haemopat win 1.0) will he introduced allowing for easier handling of data and their evaluation. this program will be used as of december 1995. in combination with a substitution calender to be filled in by all patients, in which factor concentrates, lot numbers, dosage, and date of administration will he constantly recorded, this program will extend our existing database in order to follow closely clinical and orthopedic parameters of each patient, and consequently acts as strict control of therapy quality. additionally, it provides sufficient data to fulfil any documentation needs, requested by medical authorities. the program will be available for all those interested free of charge. 2) kinderklinik der westf. wilhelms univ. mttuster 3-6) biotest pharma gmbh, dreieich haemoctin® sdh; the fviii sdh (sdh = solvent detergent and dry heat = 100 °c, 30 rain) from biotest pharma is a high purity (specific activity ~ 100) fviii concentrate manufactured from large human plasma pools. virus validation studies have shown virus inactivation/reduction (log 10) during the manufacturing process for lipid coated vints~ such as: h]v-1 > 16.2; psr > 16.8; vsv > 14,5; bvdv > 15.7; hcv > 4.5* and non enveloped vimsas such as: parvo** = 2.7; reo > 5.3*** and hav > 13.9. more than 50 hemophilia a patients (ptps = previously treated patients), baseline fviii activity < 1%, were included in an international drug monitoring study to follow their fviii inhibitur status. the hemophilia centers included were three centers from hungaria (helm pal children hospital and the national inst. of haematology, budapest and regional blood transfusion center, debrecen) and four centers from germany (two from berlin, one fraukfurffmain and one monster). patients were enrolled in the drug monitoring beginning aug. 1993. at the entry none of the patients had a detectable inhibitor. at the end of sept. 1995 there were no side effects or adverse events in connection with the use of haemuetin®. before the haemoctin drug monitoring study, the patients were treated with cryoprecipitate, or purified fviii products. inhibitor testing was done on patients plasma samples using the bethesda method. repeated fviii recovery determination at one time (between 12 to 24 hrs) after haemoctin® application demonstrated the expected recovery and normal half life time. none of the hemophilia a patients, treated with haemuetin® sdh developed a clinical relevant inhibitor. at the beginning of the stud)', the clinical efficacy of haemuetin® was studied in 16 hemophilia a patients and shown to give an in vivo recovery of 71 + 15 % by one stage assay and 77 + 17 % by a chromogenic assay. t ½ values were 13 + 2.8 and 12.7 + 3.2 hrs respectively. the study for the clinical efficacy of haemoctin® sdh was repeated in a group of 6 patients approximately two years later. although cd4 lymphocyte counts are known as reasonable predictors of prognosis in hiv infection, the cd4 count is not in all cases an infallible indicator of prognosis. therefore several serological markers are used to predict disease outcome, including beta-2 microglobulin (132m), immunoglobulin a (iga), lymphocyte counts (lymph) and others. in this study we followed a cohort of 23 haemophiliacs (19 with haemophilia a, 4 with haemophilia b) and 2 patients with severe von willebrands disease over a period of 28 months (mean, range: 22-34). testing for l~2m, igg, iga, igm, cd4 and cd8 cell counts (abs. and relat.), cd4/cd8 ratio, and absolute resp. relative leucocyte and lymphocyte counts was performed at least 3 times a year. at the same time clinical examinations and review of history were undertaken. mean of laboratory tests for every quarter of a year and significant changes during time of observation were calculated and correlated with clinical data. 1-4 5-8 9-12 13-16 17-20 21-24 cd41 440+956 344+924 278+925 302.-1:240 273.+.220 166+125 cd8 ~ 1165+474 1171+523 1236+1187 1017+439 930±412 873+478 1~2m z 2.0+0.6 3.0+1.0 3.0+1.2 3.0+1.1 3.5±1.0 3.5±1.3 lymph ~ 1.98+0.6 1.83+0.6 1.72+0.7 1.67±0.6 1.46±0.6 1.31±0.5 means/pl ± standard deviation means mg/l ± standard deviation during time ef observation we found significant changes of cd4 (abs. and relat.), abs. cd8 counts, cd4/cd8 ratio, f~2m, leucocytes and lymphocytes. the abs. cd4 and cd8 counts correlated clearly with lymphocytes und leucocytes counts but not with 1~2m. the prognostic value of the tested parameters is discussed by calculation of correlations with clinical data, anti-retroviral treatment and treatment of haemophilia. the availability of high purity factor concentrates has recently encouraged clinicians to use perioperative continuous infusion of fviii or fix to prevent or reduce bleeding in patients with haemophilia. in conliast to repeated highdose bolus injections, the continuous infusion trealment regime maintains constant coagulation factor activity at a level necessary for hemostasis, reducing the total cost of treatment by about 20% and preventing possible side effects of bolus doses. the new application mode, however, requires stable products which tolerate slow passage through an infusion device. our objective was to test in vitro the fviii concentrate immunate (stim plus) and the fix concentrate immi.ynine (stim plus) at room temperature, under conditions of long-term contact with polypropylene tubing in an infusion pump. infusion rates were chosen to mimic clinical situation. the control samples were not infused through the pump but were otherwise treated identically. test samples were drawn before and at 1, 4, 8, 24 and 48 hours after the onset of each infusion run. fviii (one-stage, two-stage and chromogenic assay) and fix (one-stage) activity were measured using immuno reagents. presence of activated factors were measured by napt'i', while flla, fxa, plasmin and pre-kallikrein activator were detected with specific chromogenic substrates. the data showed equivalent results between test and control samples with no loss of fviii or fix activity. the potencies of both immunate (stim plus) and immunine (stim plus) remained within 100 + 20% of labeued values within 48 hours after onset of infusion. in conclusion, immunate (stim plus) and immunine (st1m plus) are suitable for contiuous infusion when using automatic infusion device within applied test criteria. in htanans, circulating half-lives of asparaginase enzymes from e. coli and erwinia chrysanthemi vary within a wide range. moreover, half-lives differ not only among different e. coli strains but also among commercial e. coli preparations. to investigate the possible influence of two different sources of e. coil asparagmase (asn) preparations on the fibfinolytic system of leukemic children a prospective randomized study was performed correlating asn pharmacokiuetics (asn activity, asparagine depletion) with fibrinolytic parameters (plasminogen (plas), o.2-antiphismin (ct2ap), tissue-type plasminogen activator (t-pa), tissue type plasminogen activator inhibitor 1 (pal 1), d -i)imer (i)-d)). together with prednisono, vincristine and an anthracycline 20 children received i0000 iu-/m 2 asn medae r (originally purchased: kyowa hakko, kyogo japan) and 20 children 10000 iu/m 2 crasintin r (bayer, leverkusen, germany). blood samples for pharmacokinetic and coagulation analysis were drawn before the first asn administration and every third day whilst on medication. the results are shown in the 0.05 asn activity shows a negative correlation (spearman: rho/p) to plas (-,637/0.0003) and ct2ap (-, 751/0.0001). a positive correlation was found between asn activity and d -dimer formation (0.475/0.01). t-pa and pal 1 showed no relationship to asn activity. all children showed complete aspamgiue depletion at a detection limit of 0.1 um during the course of asn admiatstration. two thrombotic events occurred in the kyowa group, one of the distinctions between the two e. coli asn preparations administered ill this stndy is the absence of cystine in the kyowa asn, which also has a lower isoelectric point and a longer half-life than the bayer type a asn. with respect to this observations this may lead to longer inhibition of protein synthesis, which then may be the cause of a bigher rate of side effects. along with studies on asn pharmacokinoties dose recommendations need to be tailored to the specific asn preparation employed to ensure optimal antineoplastic efficacy while minimizing the hazard of complications. different types of coagulopathy in hepatic veno-occlusive disease (vod) and capillary leakage syn-drome (cls) after bone marrow transplantation w. ntimberger, s. eckhof-donovan, st. burdaeh and u. g6bel department for pediatric hematology and oncology, heinrich heine university medical center, diisseldoff, germany it is generally accepted, that cls, coagulation activation and refractoriness to platelet transfusions are part of the syndrome of hepatic vod. we assessed patients with either vod or cls or both vod and cls, in order to analyze the influence of either syndrome on different aspects of hemostasis. vod was diagnosed according to jones et al. [transplantation 44 (1987) 778]. diagnosis of cls was >_3% increase of body weight in the past 24 hours and non-responsiveness to furosemide [niirnberger et al., ann hematol 17 (1993) 67] . patients with vod, cls or both were compared to control patients without either diagnosis. eight patients suffered from both vod and cls, 5 patients only from vod, and 8 only from cls. 61 patients had neither syndrome and served as control population. activation of the coagulation system was assessed by increase of tat-complexes and/or increased consumption of at iil the hemostasis patterns were as follows: no. introduction: lung cancer goes along with coagulation activation and increased thromboembolic risk. acute phase reaction in cancer patients leads to elevated levels of c4b-binding protein (c4b-bp) followed by a shift from free to c4b-bp-bound protein s. we tried to find out whether there is a correlation between alterations of c4b-bp, protein c protein s system and interleukin 6 (il-6), which is one of the most potent inducers of hepatic acute phase reaction. patients: i. 25 patients with lung cancer; 2. control group: 11 patients in complete remission after lung cancer. methods: clotting methods: protein c and s activity; elisa tests: protein c antigen, tat-complexes, prothrombin fragments f i+2, il-6. electroimmuno-diffusion (laurell): free and total protein s, c4b-bp. results: tat-complexes and f i+2 were elevated in cancer patients. c4b-bp levels were slighthly increased (129±19 % of n.), protein s activity was 89±33 % of n. (control group: 108±23 % of n.). il-6 in lung cancer patients was 37.2252.9 pg/l (control: 27.2±8.2 pg/l). conclusion: one source of the hypercoagulable state in lung cancer patients is decreased protein s activity due to elevated c4b-bp levels. this is probably caused by hepatic acute phase reaction which is triggered by increased il-6 levels. these plasma levels correlate with levels of the tumor marker ca 125 and with the stage of the disease but correlations with patient outcome (disease recurrence and overall survival) have not previously been shown. plasma levels of d -dimer and ca 125 (determined by sandwich elisa assays} were measured prior to treatment in 36 women with figo stage t to iii ovarian cancer and correlated with tumor stage, relapse and overall survival over a mean follow -up period of 28 months (range 16 to 40 months). levels in 71 healthy women and 27 patients with benign ovarian disease served as controls. the occurrence of deep vein thrombosis in the cancer patients was also determined by impedance plethysmography that, when positive , was confirmed by contrast venography. preoperative d -dimer and ca i25 levels in ovarian cancer patients were statistically signfficantly higher than in controls. preoperative cut off values were calculated for the prediction of cancer relapse and survival for both measurements. d -dimer levels above a cut off level of 2060 ng/ml were statistically significantly associated with the rate of relapse but ca 125 levels were not. deep venous thrombosis occurred in 33 % of cases but there was no difference between properafive levels of d -dimer in patients who subsequently did versus did not develop deep vein thrombosis. high levels of d -dimer are associated with more advanced disease and with poor prognosis in patients with ovarian cancer. the high levels of d -dimer are a biologic feature of the malignancy itself that may be attributable, at least in part, to increased conversion of fibrinogen to fibrin in the tumor bed with subsequent degradation of fibrin by the fibrinolytic mechanism. thus d -dimer levels may serve as a marker for overall tumor burden as well as "disease activity". a high incidence of deep vein thrombosis exists in the course of the disease in ovarian cancer patients but preoperative levels of d -dimer are not predictive of this occurence. yon tempelhoff georg -friedrich, michael dietrich, dirk schneider, lothar heilmann. dept. obstet. gynecol. city hospital of ruesselsheim. -germany. an increase of plasminogen activator inhibitor activity (pai act.) in the plasma of cancer patients has been recently discribed. we have longitudinally investigated pai act. in 136 patients with primary breast cancer and compared the results with the outcome of malignancy. patients with untreated primary breast cancer and without proof of metastasis (t 1-4 n0-2 m0) were eligible for this study. in all patients coagulation tests including fibrinogen {method according to clauss), d -dimer (elisa} and pal act. (upa dependent inhibition test) were performed prior to primary operation, 6 months thereafter and at the time of cancer relapse. seventy -two healthy women and 43 patients with benign breast disease served as controls. during a mean follow -up of 32 + 16 months 34 patients (25 %) developed cancer recurrence and 13 (9.6 %) patients died. in all cancer patients preoperative levels of fibrinogen and pai act. were significantly higher compared to healthy women and to patients with benign breast disease. preoperatively only pal act. was significantly higher in patients with vs. without cancer recurrence (4.52 _+ 1.67 u/ml vs. 3.4 1 + 1.55 u/ml; p = 0.002). in patients with later recurrence pai a~t. significantly dropped 6 months after operation (p = 0.02) and was again significantly increased at the time of cancer recurrence (4.90 _+ 2.89; p = 0.001). a preoperative cut off value (calculated via cox model) of pai act. above 3.52 u/ml was significantly associated with the rate of relapse (tog rank: p = 0.0005) and in 70 % of patients who died of cancer preoperative pai act. were also above this cut off. impaired fibrinolysis in patients with breast cancer is significantly associated with the outcome of cancer. a monoclonal heparin antibody (mab) has been raised against native heparin using a heparin-bovine serum albumin conjugate prepared by reductive amination. for further analyses tyramine, which was covalently bound to low molecular mass heparin by endp0int attachment (malsch r et al: anal biochem 1994; 217: 255-264) , was labeled with 125-iodine at the aryl residue. the tracer antibody complex was immunoprecipitated by goat anti-mouse immunoglobuline igg. the mab recognized specifically intact heparin and heparin fractions. the lower detection limit of heparin preparations was 100 ng/ml. no cross reactivity of the mab occurred with other glycosaminoglycans such as heparan sulfate, dermatan sulfate, chondroitin sulfate a and c. oversulfated heparin showed lower affinity to the antibody hl.18 than 2-0-and 6-0-desulfated beparin. the method established for the purification of the mab was ammonium sulfate precipitation with followed dialysis. sds-page and high pressure capillary electrophoresis prooved the high purity of the received antibody. the biological activity of mab was tested by the chromogenic assay $2222 and remained stabile while purified. in conclusion, the present abstract describes an purified igg 1 monoclonal antibody directed against heparin and heparin fractions, which can be used for biological measurements. the concentration of heparin and dermatan sulfate in biological fluids is usually measured using radiolabeling. for this purpose aromatic compounds are usually used to insert radioactive iodine labeling at the saccharide backbone of the glycosaminoglycan. we developed methods for the specific labeling of hepann and dermatan sulfate at the terminal residue. tyramine was bound by reductive amination to the 2,5 anhydromannitosyl end of heparin, produced by nitrous acid degradation and confirmed by 13c.nm r spectroscopy. (anal biochem 217: 254-264, 1994) this method was also used to produce a low molecular mass dermatan sulfate (lmmd)derivative after partial deacatylation. in order to choose the proper method for evaluating the specific anticoagulant activity in the row of chitosan polysulphate (cp) samples with different degrees of pol3~merization and sulphation we applied to pharmaeapea article (a~) when assessing the ability of direct anticoagulants to depress the coagulability of recalcificated sheep blood (using the 3rd international heparin standard), and to measuring such acti¢ity as per pharmacokinetic model (a2). the model admits the "kinetics of cp elimination be linear in ease of intravenous injection to rabbits, as it is observed in heparin: ct=co exp(-i~ x t), where ct is cp concentration at the time moment t; co is cp concentration at the moment of injection; i~ is the elimination constant. besides, it is assumed that there is a linear approximation of the anticoagulant effect on the dose, which finally makes it possible to calculate the specific actidty a2 : t=kt ct + tin, where t is the time of clot formation at different tlme intervals after of cp injection; t~, is the time of clot formation prior to cp injection. t value was assessed in two tests: in blood coagulation time (bct) and in activated partial thromboplastin time (aptt). no correlation was observed between a1 and a2. at the same time the values of ifm and the period of semieliminatinn (tvz) with the use of the original method that were obtained with the help of the quantitative determination of cp in rabbit's blood taken at different time intervals after injection, showed a close correlation (1"=0,94 p<0,05) between the same parameters, obtained with the help of the of the pharmacokinetic model in bct test. thus, experimentally it was proved that the assumption of the linear elimination and the effect-dose dependence was true, which is necessary for a2 calculation. we recommend to use intravenous injection of the samples to animals with further assessment of the results according to the pliarmacokinetic model to calculate the specific anticoagulant activity in the row of chemically related potential direct anticoagulants. in this investigation we compared the biological activity of a low-molecular-heparm (lmw-heparin, mono embolcx®) after intravenous, subcutaneous and oral application in rats. sprague-dawly rats were anaesthetized by ketamine/diazepam and the blood samples were taken from the retro orbital sinuus. 150 axa u/kg body weight of the lmw-heparin were injected intravenously and subcutaneously to 10 rats each. between 3 minutes and 10 hours after injection serial blood samples were taken. 200 mg/kg (20.000 axa u/kg) body weight of the lmw-heparin were applicated orally using a stomach tube. blood samples were taken between 1 and 24 hours after oral application. the antifactor xa and antithrombin activities of the plasma samples were measured, using ehromogenic assays and the substances s 2222 and s 2238 (kabi vitmm). after i.v. injection the maximum axa and alia activities were 2.8 axa u/ml and 0.8 aiia u/nil respectively. after s.c. application the antifactor xa activity of the lmw-heparin showed a maximum of 0.5 axa u/ml atter 120 minutes. the antithrombin activity exhibited an eatiier maximum activity of 0.2 alia u/nil 60 minutes after injection. after the oral application no increase of the axa or alia activities was measured. the lmw-heparin has a high antifaetor xa and antithrombin activity after i.v. and s.c. injection. after oral application no activity of the lmw-heparin was measurable. these results implicate that fractionated heparin is not absorbed after oral application or is inactivated in the gastrointestinal tract. to improve the activity after oral application modified hepatins have to be synthesized. in an in vitro study the effect of various heparin derivatives (calciparin, fraxiparin, cy 222, cy 231, astenose, hexasaccharide, ssh 14) on thrombin-and adp-induced platelet aggregation as well as on adpmediated platelet activation in whole blood was investigated. all heparin derivatives caused a concentration-dependent inhibition of thrombin-induced aggregation of washed platelets. calciparin and astenose were found to be the most effective compounds with ic5o values of 0.67 and 1.2 p, mol/l, resp.; higher concentrations (5-30 times) were required for the other compounds. furthermore, the heparin derivatives were studied with regard to their potentiating effect on adp-induced platelet aggregation. in a concenwation range from 1 to 10 u/nil calciparin, fraxiparin, cy 222 and astenose led to a potentiation of the adpinduced aggregation whereas cy 231, hexasaccharide and ssh 14 did not show this effect. the increase in aggregation was associated with an increase in thromboxane a2 lbrmation. in addition, the effect of calciparin, fraxiparin, cy 222 and astenose on adp-induced platelet activation in whole blood was investigated by flow cytometric analysis using monoelonal antibodies to platelet surface receptors opiiia (cd-61) and p-selectin (cd-62). at concentrations that caused a maximum potentiation of adp-induced platelet aggregation these substances led to a strong increase of adp-mediated activation of platelets in whole blood. the effect was most pronounced when the blood was anticoagulated with calciparin and astenose, resp. in conclusion, the results suggest that the aggregation-promoting effect of heparin derivatives included in this study is dependent on the molecular weight and the degree of sulfation and is in part due to the generation of thromboxane. heparins are negatively charged polysaccharides and bind protamine forming a stable complex. here we report on the properties of microbeads (4.5 pro) coated by protamine. protamine chloride (0.16 ijm) was covalently bound to 0.5 mg paramagnetic tosyl..activated microbeads m-450 (dynal). the covalent binding of protamine was from 1.0 to 13,0 mg/g beads. protamine-dynabeads were produced in a phosphate buffer at different ph (7,0; 7,5; 8,0 and 8,5). the protamine-dynabeads produced ph 7.5 showed the best properties for flow cytometry analysis. in saline solution they bound lmm-heparin-tyramine-fitc (lmmh-tyr-fitc) dose dependently from 0.001 to 2 u/ml, whereas in plasma and blood they bound lmmh-tyr-fitc from 0.05 to 2 u/ml. dependent on the binding protocol, the microbeads also bind proteins unspecifically, i.e bovine serum albumine and protamine to a lower extent.the adsorbed proteins, however do not bind lmmh-tyr-fitc dose dependently. the saturation of the proteins on the beads was determined as their relative fluorescence intensity (rfi). in saline solution the saturation was measured at 380 rfi, in human plasma at 325 rfi and in whole blood at 252 rfi. using flow cytometry erythrocyctes, lymphocytes, monocytes and granulocytes were not bound to protamine dynabeads. these data demonstrate that protamine-dynabeads can be used to measure the concentration of lmmh-tyr-fitc in saline solution, plasma and blood because they do not bind to human blood cells. the present study was designed to investigate the anticoagulant action of inhaled low molecular weight (lmw)-heparin in healthy volunteers. 3,000 iu (group t), 9,000 iu (group 2), 27,000 iu (group 3) or 54,000 iu (group 4) lmw-heparin were given to 20 healthy volunteers each at 4 weeks intervals. in group 1 tissue iactor pathway inhibitor (tfpi) antigen and activity, 82222 chromogenic factor xa assay, heptest, aptt and thrombin clotting time (tot) remained unchanged during the 10 days observation period. in group 2 tfpi antigen and activity, aptt, tct and the $2222 method remained uneffected. heptest coagulation times were 18.7 + 2.0 before, 26.1 + 5.2 sec. 6 hrs and to 20.5 + 1.9 sec. 24 hrs after inhalation. in group 3 tfpi antigen increased from 74.1 + 13.9 to 80.5 + 14.2 ng/ml 3 hrs after inhalation. tfpi activity remained unchanged. $2222 method increased from 0.01 to 0.08 + 0.06 iu/ml 6 hrs after inhalation. heptest coagulation values were prolonged up to 42 _+ 7.6 s ec after 6 hrs and returned to normal within 72 hrs after inhalation. aptt and tct remained unchanged. after inhalation of 54,000 iu lmw-heparin, the following changes were observed: tfpi antigen increased to 103 +_. 17.9 ng/ml and normalized within 24 hrs. -i'fpi activity increased to 1.14 _+ 0.23 u 3 hrs after inhalation and was normal after 24 hrs. antifactor xa activity, as measured by s2222 method, increased to 0.343 + 0.196 u/ml after 6 hrs and was normal after 72 hrs. heptest coagulation values increased to 77.5 + 11.8 sec 6 hrs after inhalation and normalized after 144 hrs. aptt and tct did not change throughout the observation period. the data demonstrate a resorption of lmw-heparin by intrapulmonary route in man. no side effects were observed. recently we developed a tritium-labelled arachidonic acid ([3h]aa) release test with high sensitivity to membrane-toxic agents. the assay performed in u937 cells is intended to evaluate ehemicals, drugs and biomatefials with regard to their eytomembrane toxicity [kloeking et at. (1994) , toxicology in vitro 8, 775-777]. local irritation reactions are described in patients receiving therapeurieat dosages of lmw heparin. this fact prompted us to examine the following lmw hepafins and heparinoids for their membrane toxicity in u937 cells: reviparin-sodium, enoxaparine-sodium, mueopolysccharide polysulphate (mps), pentosan polysulfate sodium (pps), polysulfated bis-lactobionic acid amide derivatives lw10082 (aprosulate) and lw10086. for this purpose, [31--1]aa labelled u937 ceils were incubated with different concentrations of lmw heparins and heparinoids at 37°c for 1 hour. compared with untreated cells, the [~h]aa release of cells treated with 5 mg of the drugs was two times higher with reviparin sodium, three rimes higher with bis-lactobionic acid amide lw10086, five times higher with pentosan polysulfate, 20 times higher with ertoxaparine-sodlum, but it was equal to the control with mucopolysaccharide polysulphate. the rate of araehidonic acid release in response to a test chemical may therefore be used to assess the membrane-toxic effect of this substance and to predict its the inflammatory potential in the skin. semi-synthetic glyensaminoglycans (gags) with antithrombotic properties can be prepared from the e. coli k5 polysaecharide by coupled chemical and enzymatic methods. the molecular weight of these semi-synthetic gags can be adjusted to obtain products mimicking the molecular profile of a low molecular weight hepatm. in order to compare the biochemical and pharmacologic properties of a semi-synthetic gag (sr 80486a, sanofi/choay) with a commereiany available low molecular weight heparin, fraxiparine (sanofi, paris, france), valid biooheanical and pharmacologic methods were used. the molecular profile of this agent as determined by hplc exhibited a comparable distribution profile (mr=5.7 kda) in comparison to fraxiparine (ma=5.1 kda) . the anticoagulant properties of sr 80486a were comparable to fraxiparine in the aptt and heptest. however, in the usp assay, this agent showed slightly weaker activity. sr 80486a also exhibi~d comparable affinity to atffl and hcii. in comparison to fraxiparine, it produced a much weaker response in the hit screening system. in~ viv0 studies, sr 80486a preduecd strong dose-dependent antithrombotic actions in both the iv and sc studies in the rabbit jugular vein stasis thrombosis model (ed50=i 5-60 gg/kg). additionally, it also produced antithrombotic aefiorts in a rat jugular vein clamping model. the hemorrhagic effects of this agent were comparable to those of fraxipafine as measured in a rabbit ear blood loss model. intravenous administration of sr 80486a also revealed a comparable pharmaeokinetie behavior to fraxiparine. no abnomaiitias of the clinical chemistry (change in liver enzymes) and hematology profile (thrombocytopenia and lencecytosis, etc.) were noted in primates. at a dosage of i and 2.5 mg/kg iv, this agent also caused a release of functional tfpi which was comparable to the observed responses of other low molecular weight heparins. these studies suggest that sr 80486a is capable of producing similar pharmacologic effects as other low molecular weight heparms, however, additional optimization studies are required for demonslrating product equivalence. limited information on the comparative pharmacoldnetics of low molecular weight heparin (lmwh) is available on the data obtained from aptt, heptest, anti-xa and antmia assays. since these drugs are currently used for therapeutic indications using relatively high dosages and intravenous administration. aptt, heptest and antmia test may be valuable in the assessment of their effects. in order to investigate the relative pharmacokinetics of lmwh using apt'i', heptest, anti-xa and anti-iia methods, certoparin (sandoz, basel, switzerland) was administered to individual groups of healthy male volunteers (52-70 kg) via intravenous (30 mg) and subcutaneous (50 nag) routes in a crossover study. blood samples were drawn at 0, 5, 15, 30, 60, 90, 180, 360, 540 and 720 minutes. using a baseline pool plasma obtained from the same volunteers, calibration curves for each of the individual tests were constructed to extrapolate circulating levels of certoparin. a non-compartmental model using trapezoidal technique was used to obtain pharmacokinetic parameters such as t 1/2, vd, and clsys. in the intravenous studies, the t 1/2 was found to be dosedependent for aptt, heptest, anti-xa and antm]a. the auc, however, was significantly different for each test and was dose-dependent following the order: aptt<heptest<anti-xa<anti-iia. in the subcutaneous studies, 1the t 1/2 was both test and dose-dependent. the auc was also dose-dependent and followed the order: anti-xa>heptest>aptt>antmia. the clsys of the antma was much faster in comparison to the other tests. the clsys of the aptt and heptest was independent of dose. however, anti-xa clsys by this route was lower than other tests. the apparent vd followed the order aptt>antmia>heptest>anti-xa. the bioavailability of the certoparin as measured by various tests ranged from 81-119%. these studies suggest that beside providing pharmacokinetic data, aptt, heptest and anti-iia assays may provide useful data on thier safety and efficacy at high dosages. the immunological type of heparin associated thrombocytopenla (hat ii) is a severe complication of heparin treatment and is associated with arterial and venous thrombosis. only patients with absolute thrombocytopenia have prompted suspicion of hat in clinical practice. we report on a 44 year old male, who developed thromboembolic episodes after coronary angiography like reinfarction and thrombotic episodes of a. brachialis. fibrinolytic therapy combined with i.v. unffactionated heparin treatment was the therapy of choice and was followed by severe fua~er thromboembolic adverse effects. besides an impaired fibrinolytic response and elevated antiphospholipid anitbodies, we diagnosed hat type ii in hipa and elisa (stago-boehringer, marmheim). this special patient had platelet counts within a normal range, when developing the thromboembolic episodes. it appears that the normal platelet count during the thromboembolic episodes reflect a relative thrombocytopenia. from a clinical point of view we recommend the use of a lab panel to exclude hat type ii in patients with thromboembolic episodes under therapy with fractionated or unfractionated hepafin. platelet counts within a normal range are no absolute exclusion criterion for hat ii. low molecular weight heparins (lmwhs) are now commonly used for the prophylaxis of post-surgical thromboembolic complications. in this indication, lmwhs are administered as a single or twice a day subcutaneous regimen. usually these agents are administered at 30-40 mg total dose which is equal to 3000-4000 anti-xa (axa) iu. newer methods such as ehromogenic substrate based axa methods and the heptest clotting time can be used to determine the effects of lmwhs during the initial phases of prophylactic therapy. this may be useful in the elderly and weight compromised patients where a fixed dosage may not be optimal and may produce bleeding effects. similarly in the overweight patients, a fixed dose may not be efficacious. thus, monitoring of lmwhs in these patients may be useful in the optimization of their therapy. lmwhs are also used in the treatment of deep vein thrombosis using both intravenous and subcutaneous protocols. high dosages of up to 200 mg sc/day and infusions of up to 30 axa iu/kg/hr have been administered. in these conditions, the monitoring of the circulating lmwh levels may be useful in optimizing the dosage. we have modified the aca heparin (do_pont merck, wilmington, de) method to measure the lmwh levels in the plasma of patients treated with both the prophylactic and therapeutic dosage. owing to the required turnaround time, simple operation and reliable results, this method was found to be of value in the monitoring of these agents. this presentation provides an overview of the clinical application of various lmwhs with particular reference to the need of monitoring for their effects to optimize the clinical outcome. a double-blind, multicentric, controlled trial was performed in order to compare the antithrombotic efficacy and safety of single daily doses of 5000 ie anti-xa of low molecular weight heparin (lmwh) sandoz (certoparin) and 5000 ie unfractionated heparin (ufh) tid. in 288 patients undergoing elective total hip replacement blood samples were drawn before the first subcutaneous injection of lmwh or ufh resp., two hours after administration on the first and 7th postop, day and on the last day of prophylaxis (day 10-14), anti-xaactivity was measured by chromogenic substrate assay, heptest and aptt by clotting assays and tissue factor pathway inhibitor (tfpi) and heparin-pf4-antibodies by elisa techiques. as expected, the anti-xa-activity and the heptest values were significantly higher in the lmwh-group at all time points after administration of the drugs; the mean values of heptest were 35 sec in the ufh-and 75 sec in the lmwh-group respectively, the aptt was not different in both groups. at the end of prophylaxis positive antibodies to heparin-pf4complexes were detected ~n both groups; this however was not correlated with clinical thrombocytopenia. a detailed correlation between patients with deep vein thrombosis (dvt) and positive antibodies has still to be done (all patients were screened for asymptomatic dvt between day 10-14 by bilateral phlebography. tfpi was markedly increased in the lmwh-and only slightly elevated in the ufh-group; the differences are statistically significant. summarizing it can be concluded that antibodies to heparin-pf4complexes may occur without clinical symptoms of hepafin-induced thrombocytopenia type ii and that tfpi may play a sigificant role for the antithrombotic efficacy of ufh and lmwh. unfractienated heparin represents one of the most severe and frequent causes of drug-induced thrombooytopenia. heparin-indueed thrombocytopeala (hit) occurring early in therapy is often mild and serf-limited, appearing to be caused by a direct aggregant effect of heparin on platelets (hit type i). hit type ii, however, is immune-related an may result in absolute thrombocytopenia (platelet count <i50/~l) or a relative decrease in platelet count (?_50% reduction from baseline values). unlike most other immune thrombecytopenias in which bleeding complications often predominate, hit type ii may results in severe arterial and/or venous thromboses. the management of patients with hit type ii and thrombosis is often difficult. heparin must be stopped immediately. antithrombotic, fibfinolytic, or antiplatelet agents have been used in these situations, as have anticoagulants other than unfraetionated hepafirl plasmapheresis, used in other immune-related disorders, has also been occasionally reported. at our institution this intervention appears to provide significant elininal benefit to hit patients. several recent reports have noted that the heparin-dependent antibodies in hit type ii are directed against a complex of heparin bound to platelet factor 4 (pf4) and an elisa-based test system for hepafin-pf4 eomplexinduced antibodies (hpia; stago) has been develuped~ we report our experience in 3 patients with hit type ii in whom plasmapheresis was instituted and the antibody levels in blood were measured pre-and post-treatment to identify wkether circulating antibody titers decreased thus providing some explanation for the clinical benefit. the patients developed profound absolute thrombocytopenia (range, 3-21 k/~tl) and positive heparin-induced platetet aggregation studies following treamlent with uofractionated heparin. thromh0cytopenia induced by hepariu (hat-syndrome) typically appears several days after initiation of heparin therapy. this phenomenon is caused by antibodies of the lgg type which are induced by a heparin-pf 4-complex and which also activate platelets. in a recent study (n engl j med 1995; 332:1330-5) the hat-syndrom was diagnosed in 2.7 % (9 of 332) patients who had received unfractionated heparin (ufh) and in none of those 333 patients who had been treated with low-molecular-weight heparin (lmwh). 8 out of the 9 patients treated with ufh presented with at least one thrombotic event (7 times a venous, once an arterial event). the incidence of heparin-dependent igg antibodies (7.8 %) was higher in the ufh treated group than in patients treated with lmwh (2.2 %). our patient was a male caucasian white, 62 years of age, otherwise in good clinical condition with no severe diseases in his pmh. both parents, however, had suffered several thrombotic episodes. the patient was admitted to the hospital in september "95 with clinical signs of a deep vein thrombosis (dvt) of the right leg. the diagnosis was phiebographically confirmed. after having given informed consent the patient was treated with 5 cycles of r-tpa (loco-regional application) and 3 cycles of uhsk as part of a clinical trial (uhsk vs. rtpa, boehringer/m). lysis was successful with complete recanalisation of the veins. after completion of lysis the patient was further anticoagulated with coumarin overlapped by heparin (ufh). as a complication the patient suffered from a retroperitoneal and an upper gi4ract bleeding from esophagitis grade ii to hi while being on coumarin prophylaxis. therefore the patient was put on lmwh and then discharged with this prophylaxis. after less than two weeks the patient presented again to the hospital with a relapse of dvt in the same leg. phiebographically nearly all deep veins were occluded to a higher degree than before, i. e. also with concomitant pelvic vein affection and with hea~ 3, swelling of the whole leg. blood count showed a marked decrease of platelets to 27/nl. the platelet aggregation test demonstrated a hat-syndrome which was confirmed by the hipa-assay. the heparinoid orgaran® was not reactive in the assay. having seen a previous bleeding episode in this patient, we did not see an indication for another b'sis therapy. therefore we decided to put the patient on an intermediate dose of coumarin overlapped by hiradin (hat study, behring) instead of orgaran®, since hirudin allows a much more precise dose adjustment. after 10 days the patient was discharged with no further complications. risk factors for dvt have not been detected until now. to our knowledge this patient is one of the first cases of hat-syndrome induced by lmveh. under routine conditions aptt is the most frequently used test method for the surveillance of patients undergoing hepann therapy. in literature a 1,5 fold to 2,5 fold prolongation of the initial value is recommended for individual therapy a~ixatian. in contrast to the control of the enmarin therapy by means of prothrombin time measurement the individual hupafin sensitivity of the ~ reagents of this standard value hardly receives notice. commercially marketed ~ reagents differ from one another method and concentration of the activator as well as their phosphelipids. when choosing a reagent the user must only take into consideration the sensitivity against its factors, heparin and lupus but also the adalxability of the reagent towards the given measurement system ( software variability towards incubation time, sodimentalion problems in reagents with high kaolin concentration). reagents offering a high heparin sensitivity already are showing a prolongation of the coagulation time at low heparin dosage (0.1 -0.3 u/ml). in the therapeutic range (0.4 -0.8 u/ml) very often no linear relationship between prolongation of the aptr and heparin concentration level is found_ therefore very often coagulation times above the routine measuring range (180 s) can be observed in this range. moreover, various medical measuring devices differ in their ability to recogniso the retarded entry of coagulation. coagulation limes with different apq't reagents in correlation with the heparin concentration level can be found in the adjoined table. significantiy prolonged coagulation ti.~es of aptt lead to a reduction of the hel~ dose in clinical practice. due to the discrepancy between heparin concentmon level and the prolongation of the afrt in reagents with a non-linear heparin sensitivity a reduction of heparin dosage leads to an inmt~cient anticeagulalion and the risk of further thrombosis. therefore the producers should be asked to include precise directions on the heparin sensitivity of the aptt reagent in the therapeutic range in order to enable an optimal surveillance of the heparin therapy. a coordination between clinic and laboratory should be compulsory for the choosing of the aptt reagent. introduction: hat is a rare but sometimes fatal complication of therapy with unfractionated (ufh) and low molecular weight beparins (lmwh). we report on our experience with the clinical coume and management in 12 patients (pts). in all cases a severe decline of platelet count and in 11 cases a positive hipa test (= heparin-induced platelet antibodies) were documented. patients, clinical course and outcome: 8 female and 4 male patients (median age 68 years, range 22-78) were analysed in this retrospective study. 9 pts were referred from outside hospitals. all pts had been treated with ufh (5 pts additionally with lmwh) for prophylaxis (n = 9. in 2 pts pedoperatively) or treatment (n = 3) of venous thromboembolism (v'te). in 11 pts at least one vte occured during heparin therapy, in all cases prior to hat-diagnosis (vte: n = 13; putmonary embolism: n = 6; artenal embolism n = 1; thrombosis of sinus transversus n = 1). hat was suspected 16.5 days (median; range 12 -48) after first heparin application. hepadn therapy was discontinued immediately, at that time the median plateiet count was 35 g/i (range 10 -117). 9 pts were treated with orgaran r, followed by recombinant hirudin (r-hirudin, behdngwerke ag: clinical study) in 4 cases; 3 pts were treated exclusively with r-hirudin. plat~let count normalized in 9 pts within 3 days (median; range 1-10). one patient in the orgaran" group died on recurrent pulmonary embolism. 11 pts recovered without any further thromboembolic complication. conclusion: in our small series of 10ts hat was complicated with vte in 11/12 cases. in many pts hat was diagnosed strikingly late. for early diagnosis careful monitonng of platelet counts are mandatory dunng hepadn therapy, both orgaran ~ and r-hirudin seem to be safe and effective in preventing further thromboembolism, p 179 to prospectively evaluate coagulation or fibrinolytic activation in children long-term anticoagulation with lmwh was administered a longitudinal study was perfomed. within a 30 months period 48 children and adolescents (6 -19 years) suffering from malignant bone tumors (ewing's sarcoma or osteosarcoma) received enoxaparin (clexane r/rhone-poulenc rorer, france) for primary propylams. the majority of patients received 0.6 mg/kg bw enoxaparin 12 hours before major surgery (limb salavage). in addition, ten neonates and children received enoxaparin in the same dosage for secondary prophylaxis. in both groups therapy was adjusted to 0.2 -0.4 iu/ml anti xa activity. median (range) duration of therapy was performed for l0 (6 -24) weeks. before ('1"1), one (t2) and 3 weeks (t3) within the context of the present study an investigation on the physiology of clotting was conducted in three groups each consisting of 12 patients, who had to undergo a total end-prosthetic hip implantation (teph) for the first time. criterion for allocation to a particular group was type of intraoperative transfusion the patient received : infusion of whole blood, erythrocyte concentrate or fresh frozen plasma. at eight fixed times the activity or concentration of the components of the kallikrein-kinin and inhibitory systems was determined. the statistic evaluation ensued with the spss software package. for all three patient collectives an activation of both systems was to be observed with the teph-implantation whereby there were no statistically striking differences between the three groups. thus in the case of the kallikrein-like activity an intraoperative rise of over 37% was registered, while at the same point in time there was only a 4% reduction in the prekallikrein activity. postoperatively a 10% rise in the prekallikrein activity was to be observed. as a reserve inhibitor alpha 2-macroglobalin showed no significant change in activity during the period of observation whereas the acute phase proteins alpha 1-proteinase inhibitor and cl-esterase-inhibitor yielded a postoperative rise of 135% and up to 100%. the beta-factor xllainhibition showed a rise of 121% during the reconvalescent phase. the 65 year old female caucasian patient presented here, was admitted to a nearby district hospital in december '94 with the acute symptoms of pulmonary embolism. the source of the embolus could not be detected. high dose heparin was administered. pmi-i revealed a coronary artery disease and a peripheral artery occlusive disease of the left lower leg, yet blood pressure values of the lower ex~emities were only slightly below brachial pressures. the first episode of pulmonary embolism was followed by a second one two weeks later for which the patient received heparin again. following this treatment the platelet count decreased significantly to 10/ni. with an acute thrombosis of the lower caval vein and with leriche's syndrome, i. e. occlusion of the abdominal antra from the bifurcation down to the ileofemoral arteries bilaterally, the patient was transferred to our hospital. on initial examination we saw a patient in no acute distress, no dyspnea, blood pressure 120/80 mmhg, pulse 94/rain, no peripheral edema or disturbances of sensitivity. neither femoral, popliteal, nor pedic pulses were palpable on either side. a heparin associated thrombopenia was detected with the platelet aggregation assay and confirmed by the heparin induced platelet aggregation assay (hipaa). the autoantibodies targeting the underlying pf4-heparin complex were determined later by an elisa method in serum samples of the patient (asserachrom hia, diagnostica st,ago). a temporary anth&~r filter system was inserted transbmchially into the lower eaval vein and placed jnst above the thrombus. in the following days, 2 cycles of lysis therapy with 9 mil u streptokinase were administered taking six hours for each cycle. heparin was given in the mean time via the filter system in therapeutic dosages in order to prolong values 1.5 to 2 times the individual base line level. at the end of the second cycle, pulses were palpable inguinally, two hours later both popliteal and the right pedic pulse were present. the left leg showed better microperfusion than before. computer tumoglaphy did not reveal an)-thrombotic material in the descending aorta and the iliac arteries. for continuous anticoagulation after lysis we used the heparinoid orgaran® to overlap the period until full anticoagulation was achieved by coumarin. the heparinoid was tested before us non-reactlve in the hipaa. with this prophylaxis the platelet count increased to normal values. hat ii related thrombosis is the most severe complication of heparin therapy, especially in patients with pre-existing thromoembolic disorders (dvt, dic). we present to our knowledge the first case of hat ii during thrombolytic therapy (it) with simultaneous hepatin administration. at admission a 16 year old girl showed complete occlusion of the pelvic veins of the right leg (r common and external iliac v., common and proximal superficial femoral v.). as in children no prospective studies on the thrornbolysis of dvt with rt-pa are available, we started tt according to the frankfurt/m. regimen: rt-pa (actilyse) bolus injection 0.4 nag/kg bw followed by continuous infusion of 1 mg/kg bw/d; simultaneous low dose heparin 100 -200 u/kg bw/d. doppler ultrasonographie control on day nine after the beginning of tr revealed a further distal growth of the thrombus into the popliteal vein. at the same time at ir was decreased to 70% and there was a dramatic drop in platelet count to <50% of the baseline value (216 to 86 gpt/1). the diagnosis hat ]i was confirmed by positive hipa-test. the only heparin preparation not cross-reacting with the antibody was the lmw heparinoid orgaran. heparin therefore was replaced by orgaran: bolus injection 1,500 u/h followed by infusion of 200 u/h. rt-pa-therapy was not discontinued. at ri substitution was performed at levels <70%. under orgaran administration platelet count rose from 77 to 239 gpt/1 within 5 days. on day 12 complete reperfusion was achieved and rt-pa was discontinued ,on day 14. the patient was discharged from the hospital on prophylaxis with phenprocomnon in good general condition at admission thrombophilia parameters (protein c and s, apc) were within normal range. heparin induced thrombocgopenia (hit) is a serious but rare side effect of treatment with unfractionated heparin. patients with hit present with a rapid decrease in thrombocyte count and occurence of venous or arterial thrombosis under heparin treatment. patients with arterial occlusive disease who need surgical intervention are of high risk to develop rethrombosis especially during the first few postoperative days while they are under i.v. heparin-treatment. in our study we investigated prospectively all patients who were admitted to a surgical unit because of arterial occlusive disease and received surgical intervention. 15 patients were screened on the 3rd and 6th postoperative day for heparin-antiplatelet 4-antibodies, decrease in platelet count and thromboembolic complications. 3 patients developed antibodies against heparin-platelet factor 4. one of the 3 patients developed a venous thrombosis in the operated leg in combination with a decrease in thrombocyte count. we conclude that heparin-platelet factor 4-antibodies are often produced in patients under i.v. heparin treatment. only few of these patients actually suffer from clinically s3anptomatic hit. medizinische universit~itsklinik, institut far klinische biochemie und pathobiochemie, d-97080 wtirzburg, germany the important physiological and pathophysiological role of platetets in health and disease is well established. platelets are also the primary target of many vasoactive hormones, factors and drugs. in addition, platelets provide a very important model system for studying agonist-and antagonist-mediated signal transduction events (1,2). platelet activation by agonists is associated with shape change, extension of filopodia, generation of intracellular messengers, secondary agonists and activation of adhesion receptors and integrins including the fibrinogen receptor gp iib/iiia. activation of protein tyrosine ldnases and calcium-dependent protein kinases appear to be of critical importance for the mechanism of platelet activation. in contrast, platelet inhibition by vasoactive factors and drugs is often mediated by the camp and/or cgmp signal transduction cascades. recent data form our laboratory suggest that the focal adhesion protein vasp, a major target of the intracellular no/cgmp and prostacyclin/camp effector systems, is an important link between signal transduction pathways and elements controlling cell motility (3-6). the implications of the recent advances in platelet signal transduction research for understanding the physiology, pathophysiology and pharmacology of human platelets will be discussed. domain. however, in vivo truncated "i'po is much less potent than full length tpo due to its rapid clearance. this suggest that the c-terminal glycosylated domain acts to stabilize circulating tpo. tpo stimulates both proliferation of progenitor megakaryocytes and their maturation to platelet producing megakaryocytes in vitro. in vivo tpo induces dramatic increa~ses in megakaryocyte number and platelet production in animals, indicating that tpo regulates both thrombopoiesis and megakaryocytopoiesis and is the primary regulator of physiological platelet production. this later notion is supported by the phenotype of ti~ and cmpl knockout mice in which platelet levels are only 10-20% of normal while other blood lineages are unaffected. in animal models of myetosuppresion ,and marrow transplant, tpo reduces the platelet nadir seen in the~se models and significantly accelerates platelet recovery, tpo also has profound effects on red cell recovery in these models. these results suggest that tpo will be clinically u~ful for the treatment of thrombocytopenia in caucer patients who undergo chemotherapy or bone marrow transplant. the haparg-study ( .~a.emostatic parameters as risk factors in healthy volunteers) was sponsored by the german ministry of research and started in 1984. a previous study ( pard, intern. angiology 5, 181-195,1986 ) had shown that spontaneously enhanced platelet aggregation as measured with the pat-ill-test and a high fibrinogen level were significant risk factors for new vascular occlusions in diabetic patients. it was the aim of the haparg study to establish if spontaneous plateletsggregation and other hemostatic variables are independent risk factors for vascular occlusions also in healthy volunteers. employees of hschst ag, frankfurt aged 40-65 years and personnel of the university of mainz, aged 30-60 y. entered this prospective study. besides anamnestic data on smoking, hypertension and diabetes blood pressure, the ankle/arm doppler -index and an ecg were recorded and serum cholesterol, hdl, ldl, triglycerides, uric acid and glucose were measured. 1885 men and 988 women entered the study and were followed for 4-6 years. during this period 53 vascular occlusions ocoured ( 36 coronary and 9 cerebral events and 8 peripheral vascular occlusions). only three of these events occured in women. besides age and gender as expected risk factors the multivariate logistic stepwise regression analysis revealed as significant risk factors slightly elevated levels of blood glucose (p=0.001), smoking (p=0.0013), an elevated diastolic blood pressure (p=0,018),followed by spontaneous aggregation (p = 0.017) and higher hdl-levels (p=0.034) as significant risk factors for men. higher fibrinogen levels just missed significance in this analysis (p=0.059). none of the other hemostatic parameters showed any significant correlation with the vascular events.to our knowledge this has been the first prospective trial in a large population of healthy individuals in which a platelet function parameter has been studied together with other possible risk factors. spontaneously enhanced platelet aggregation seems to be an independent risk factor and may be an indicator of an ongoing active atherosclerotic process. mild bleeding symptoms -petechia, prolonged bleeding after tooth extraction -were observed in a patient with normal coagulation tests but slightly disturbed phtelet function: decreased aggregation in response to adp, pal: and thrombin receptor-derived peptide (trap-6). platelet count, agglutination with both ristocetin and botrocetin and thrombininduced aggregation were in the normal range. an abnormal membrane glycoprotein detected in electrophoretic studies of the patient's platelet proteins was shown to be related to gp llxx by immunoblotting. the variant gp differed even from the largest normal polymorphic gp iba form a by an increase in molecular mass, platelets of the patient contain the variant and normal c form in equal amounts. the quantity of gp ib as detected by flow cytometry is normal. lmmunoblotting studies of proteolytic fragments of the abnormal gp indicated that the defect is located in the c-terminal part of the glycocalicin fragment. measurement of the length of glycocalicin molecules after glycerol-spraying rotary shadowing electron microscopy demonstrated two populations of molecules in the patient sample: a normal on with an average size of 470 angstr6m and a fraction with an average size 0f650 angstr6n~ preliminary results of molecular analysis of the gp iba gene in the patient confirm the localization of the genetic defect by protein studies. in summary, the patient is heterozygous for a hitherto undescdbed molecular variant ofgp ~a. barnes and co-workers have shown that simple triple-helical collagenlike peptides, comprising basically a repeat gly-pro-hyp structure, are highly platelet reactive in polymeric form. they additionally have shown that the activity does not involve gpla/lla (integdn c~z~l). platelets adhere under static conditions to these fibrillar peptides mainly in a bivalent-cation independent manner; the bivalent cation dependent adhesion can be inhibited by an antibody against gpflb/llla (integfin m~d33). however, platelets of a patient with type i glanzmann's thrombasthenia secreted their ¢z-granule-and dense body content just like normal controls while activated with the triple-helical gly-pro-hyp peptide. the lack of an essential involvement of cd36 was shown with cd36deficient platelets, which respond to the peptides as readily as normal platelets when fibdnogen binding and cd62 and cd63 expression were measured. to study the involvement of the yon willebrand factor (vwf) platetets from a patient with severe type 3 vwd (vwf not detectable in platelets and plasma) were investigated. the vwf deficient ptatelets responded quite normally to the fibritlar collagen-like peptide. addition of pudfied vwf to the vwf-deficient platelets before adding the paptide raised the platelet respond slightly we conclude that neither ~3~ integrin, nor m~ integdn, nor cd36, nor vwf are the pdmary platelet receptor for the collagen-like peptide. there is evidence that native low density lipoprotein (ldl) can be oxidatively modified in vivo and the oxidized ldl may contribute to thrombogenesis and atherogenesis by damaging endothelial cells, eliciting vasocontractiola and stimulating blood platelets. by means of biochemical methods, electron microscopy, reflection contrast microscopy and computer image analysis, we find that oxidized ldl, compared to native ldl, affects platelets at least in the following five aspects. 1) oxidized ldl alters platelet functional morphology in a time and dose dependent manner. 2) oxidized ldl induces secretion of serotonin from both morphological resting platelets and shape changed platelets. 3) oxidized ldl advances, accelerates and enlarges platelet adhesion. 4) oxidized ldl causes cytodamage in platelets and cultured endothelial cells. 5) oxidized ldl stimulates platelets and endothelium, which leads to a formation of microthrombi on the monolayer of human umbilical vein endothelial cells. further experiments show that oxidized ldl inhibits the activity of ca2+-atpase in purified plasma membranes of platelets, and therefore increases the cytosolic calcium level in platelets. these results clearly indicate that calcium signaling pathway is intimately involved in the mechanism of platelet activation induced by oxidized ldl. since oxidized ldl, a relevant platelet agonist, has been confirmed to be present in vivo, further studies on its role in platelet activation and plateletendothelium-interaction can help for better understanding thrombogenesis and atherogenesis. recantly physical parameters such as sheer stress, hypo-or byperoxic conditions and hyperthermia have been shown to modulate endothelial cell dependent fibrinolysis. in this study we investigated whether yet another physie~ stimulus namely hypothermia might have an impact on the expression of components of the fibdnolytic system of human endothelial cells in culture. confluent cultures of human umbilical vein endothelial cells (hlivecs) were exposed to gc ° for 10, 20, 40, 80, or 160 rain, respectively. control cultures were exposed to 37°c for the same time periods. thereafter medium was removed, fresh medium was added and the cells were incubated for 24 hours at 37°c conditioned media (cm) were harvested and plasminogen activator inhibitor-i (pal-i) antigen was determined by a specific elisa. our data showed that huvecs responded to exposure to 8°c with a time-dependent increase of pai-i in cm. maximum induction of pal-! antigen was seen after 20 minutes exposure to 8°c (pal-! antigen: 754_+.83 ng/105 cells for treated cells; 4202_17 ng/105 cells for control cells). the inereese in pal-1 antigen was also reflected on the level of specific mrna synthesis as evidenced by northern blotting which showed a twofold increase in pai-i specific mrna in huvecs exposed to 8°c as compared to control cells. in conclusion, our data gives evidence that hypothermic stress like several other forms of physiological or pathological stress, results in activation of endothelial ceils as evidenced by an increased expression of pai-i. our findings provide ~rther evidence that such activation of endothelial cells can be induced not only by biochemical mediators but also by physical stimuli. nitric oxide (no) is formed from intracellular l-arginine by two no synthase (nos) isoforms: a ca+ +-dependent constitutive form (cnos) and a ca+÷-independent inducible form (inos) which is expressed after challenge of cells with immunologic or inflammatory stimuli. endothelial cell derived no is an important signaling molecule inducing smooth muscle cell relaxation and platelet inhibition, however its role in cell proliferation is poorly understood. the present study investigates the pattern of nos isoforms and no production in actively proliferating low density human umbilical vein endothelial cells (ld-huvec) and in high density monolayers of quiescent cells (hd-huvec). ld-huvec but not hd-huvec expressed inos (measured as ca ++independent citrulline formation from l-arginine in cell lysates) with a maximal activity of 30-40 pmoles/min/mg protein, cnos (ca +÷dependent citrulline formation) was demonstrated in hd-huvec (0.02-0.2 pmoles/min/mg) but not measurable in ld-huvec lysates. lmmunoprecipitation of cell lysate with a mab against murine macrophage inos decreased inos activity of supernatant by 70% in ld-huvec. immunoprecipitation with an anti-ecnos mab did not modify inos activity in ld-huvec but abolished cnos activity in hd-huvec. no release (measured as nitrite + nitrate production in an 18 h incubation in the presence of 1 mm l-arginine) was 1.9 nmol/mg cell protein in the supernatant of hd-huvec whereas it was t0fold higher with ld-huvec. measurement of intracellular cgmp after stimulation of cells with i-2 #m ionomycin (a sensitive index of enos activity) showed a 400-800% increase of basal levels in hd-huvec but only 50-150% increase in ld-huvec. on the contrary, sodium nitroprusside-induced cgmp increase was similar in non-confluent and confluent cells. the expression of inos and the high no production in ld-huvec suggest a role of endogenous no in mediating cell proliferation. indeed, the nos inhibitors l-nmma and l-canavanine inhibited [~h]thymidine incorporation in ld-huvec by 60%. on the ++ nt no r uctlon ar x r d m other hand enos and ca -depende p od " e e p esse " differentiated huvec and correspond to the ability of the cells to regulate vascular tone and platelet activation. the processes of replication, maturation and motility of progenitor cells of different lineages in the bone marrow are tightly regulated, particulady by various cellular contacts and their modulation involving pericellular proteolysis. while mononuclear phagocytes and related cell lines are well characterized for surface localized plasminogen activation by receptor bound urokinase, megakaryocytes are poorly described in this respect. in the present study the expression of mrna of urokinase receptor in megakaryoblastic cell lines chrf-280, dami and meg01 was found at a low basal level and increased 5-10 fold after pma-treatment. urokinase receptor mrna levels remained differentiation dependent elevated for up to 5 days. functional analysis of upar expression on the cell surface was documented immunologically by facs-anatysis and on the functional level by direct binding of radio-labeled amine-terminal fragment of urokjnase and mirrawed the increase in urakinase recptar mrna level. as expected, antibody and ligand binding was sensitive towards treatment with phosphatidyl-inositol-specific phospholipase c, since the urokinase receptor belongs to the group of gpi-anchored receptors. in cultered megakaryocytes derived from bone marrow urokinase receptor mrna was demonstrated by rt-pcr, as well. these results indicate a differentiation-dependent increase of urokinase receptor expression on cells of megakaryoblastic cell lineage suggesting the participation of urokinase dependent events during maturation process and intravasation of these platelet precurors. regulation of am gene expression may be coupled to oxidative stress through redox sensitive transcriptional regulatory factors. the aim of this study was to investigate the influence of selected antioxidants on tnfa (100 u/ml, 6 h)-induced surface expression of elam-i, icam-i and vcam-i in human umbilical vein endothelial cells. am expression was determined by quantitative flow cytometry and immunohistochemistry (alkaline phosphatase anti-alkaline phosphatase method). the following antioxidants were tested: pyrrolidine dithiocarbamate (pdtc), n-acetylcysteine (nac), glutathionemonoethyl ester (gshmee), probucol and the estradiol-derived scavestrogens j824, j861, j901. additionally, 17fi-estradiol was examined. the used concentrations were 1-5 mm for nac and gshmee and 1-100 ~m for all other substances. according to their influence on tnf-induced am expression, the tested antioxidants can be divided into three groups: 1) pdtc, nac and gshmee inhibit the expression of all three am, 2) j861 and j824 reduce vcam-i and icam-i and increase elam-i, 3) j901 and probucol show no effect. 17fl-estradiol elicits a potentiation of tnfa-induced expression of all three am. tnfa-induced vcam-i expression was most effectively inhibited by ptdc and j861 (100 em: 68% and 78% inhibition, respectively). the best inhibitors of icam-i induction were pdtc (100 ~m: 51% inhibition) and j824 (100 t~m: 47 % inhibition). j824 and j861 most effectively increased the elam-t induction (100/~m: 85 % and 62 %, respectively). the results confirm that antioxidant-sensitive mechanisms play a role in tnfct-induced am upregulation. it is suggested that icam-1-and vcam-l-expression is regulated differently from elam-i. the diversity of antioxidant effects might be due to distinct levels of redox control of transcription with an antioxidant-inhibited and an antioxidant-promoted step. heamophiliacs who continue to produce tilers of f.v11i inhibitors no higher than 5 to 10 bethesda units (bu) when given continuous f,viii replacement therapy are classified as low responders. studies carried out on low responders to assess the effect of immune tole~lcc therapy flit) in these patients. it was found that elimination ofiahibitors in low responders could be achieved using f.vlii at 20 to too u/kg given every 2 to 3 days over a period of 6 weeks. we consider that 1tt is justified in this group of patients. prevents rebooster effects and reduces elimimfion time of f.viii inhibitors. it also reduces the enhanced mortality in patients with inhibitors and has been shown to be cost-effective. in view of the improved prognosis, we recontmend starting itt in low respor, ders soon after the diagnosis has been made in order to minimize f.vill exposure before isfi.tiating el'. itr in low responders has a better success rate an.,d requires shorter u'eatment than itt in high responders. w.schramm, med.klin~, innenstadt, lmu, mfinchen following replacement therapie~ in hemphilm t_be developmem of inhibimrs to the p*ficat's deficient factor is the most serious sequelae. the incidence of inhibitors varies between 15 and 35 % of persons with hemophilia a. inhibitor develop, mostly in paticm~ with sovere bemoph/lia a, early in life and afmr few exposure days (9 -12 days). using the bethesdamethod the inh~itom can be derided in low (< 5 bu) and high msponder (>5 bu) hemopb~iacs with high fitcrs have ~ually serious ~ problems. they are resistent to mg,,flary replacement therapy, the ~ goal in the treawnent is to control severn acum bleedin~ and to eradicate the inlu'bitor perrmnanfly and to induce tolea'ance. in the tream'tmt of acute blcedings in patients with hlhibitors factor viii inhibitor bypassing ag~ts like activated prothro~ complex concenuxtes (feiba) or prothrombin complex concentrates (pcc) arc mostly used. the meehani~n of aefiou of theses concentrates is net fully investigated. their effect is usually related to the high coment of activated clotting factors ~d phosphoupids. since some years acdwated recombinant factor vii (f vii a) is used to treat patients with inl'dbitocs successfully in several clinical situations including surgery. in addition porcine factor vii1 is widely used in particular in the uk for the treatment of factor v].ii inhibitor patients and could demonstrade good clir3cal results, in case of life threatening bleedings a temporary reducfic~ of inhihitors could be. ~hieved by using extem*,ivc plasma exchange (protein a adsorption) and immune suppression with cyclophosphamid (~alm6 protocol). follow~g the first description by h. bmc~'~mn some modifications for tlm induction of irmnune tolerance in hemophilia a patients have ~en propet'ed. these schedule, can be derided into high, intemxxfime and low dosage roglrmms di:ffea'jng in the dosage of factor viii infused. successful rates about 70 to go % can be obtained with ~ and high dose regimens. but is has to be co~sidered that the~ expensive trea.t~nt regimens have a great physical and p .syc.hosocial impact to the benx~-li~s and thch" farm~e& the different immu~ mler-a.~ze mg~-'~ predominantly used in high rcsponder inhibitor. most of the patients with low concentrations of inhibitors cm be managed with factor viii in increased dosage. this is in agreement with the consensus recorrnr~rdadons for u'eatlncnt hemophiliacs in germany fi'orn 1994. before vitamin k(vk) prophylaxis was generally accepted in japan, the incidence of infantile vk deficiency was 1:4000 both idiopathic and secondary types. since 1981, 4 nationwide surveys have been conducted. the current incidence rate is now about one-tenth that in early 1980. however, in a small number of eases, vk deficiency oceured despite prophylactic administration during the neonatal period. in order to clarify the absorption,excretion and transplacental transport of vk in the perinatal period,following studies were carried out. t)hepaplastintest(normotest) were performed on 65 women in the last stage of pregnancy and each coagulation factor was estimated as well. 2)correlations were made between mothers'and babies'hepaplastin test values. 3)transplacental transport of vk 2 was studied. the general activity of vk dependent factors in pregnant women was much higher than in non pregnant women. as far as the correlation between mothers'venous blood during delivery and cordvenous blood is concerned, in the group of mothers with hepaplastin test value of less than 120% of the normal adult value, the value of the hepaplastintest was less than 30 % of normal adult value in the cord venous blood° we also demonstrated that vk passed through the placenta but only in small qualities. 61hiv-negative patients (median age 4]yrs, 13-70}, formerly treated with non-virusinactivated coagulation products, underwent hepatologic examination, including afp screening and sonography. 31.suffer from severe, 20 from moderate or mild haemophilia a or b, 10 from other severe coagulation factor deficiencies. 52 had been treated with products of the swiss red cross (src) only (28 with small pool cryoprecipitate}, 4 with foreign products only, 5 with both src and foreign products. treatment intensity was variable with>20'000 iu/yr in 26,< 20'000 in ]6, < 1 treatment episode/yr in ]0, a total of only 1-3 treatment courses in 6 patients. 3 afibrinogenemic patients had prophylactic replacement therapy. hcv serology was positive in 56/6] patients (92%), in 47 with detectable hcv rna (77%). the 5 persons who escaped hcv infection, with normal alt-levels and without sonographic alterations, had low intensity treatment with small pool src preparations only. alt-levels were elevated in 33/56 anti-hcv positive patients (59%). 26/56 had abnormal sonographic findings (46%). there was a clear correlation between elevated alt-levels and abnormal sonographies: of 33 patients with elevated alt 23 had abnormal sonography, of 23 with normal alt 3 had abnormal sonography. 8 patients had liver cirrhosis (6 with clinically overt hepatopathy), 4 (4/56 = 1%!) with hepatocellu]ar carcinoma (hcc) with elevated afp-leveis. of these 4 patients 2 had intraarterial embolization with ]ipiodol-epirubicin; in 2 patients hcc diagnosis was made in a late stage. i patient with advanced liver cirrhosis underwent successful liver transplantation. 2 of the 6 patients with hepatopathy had severe haemophilia with temporary high alcohol intake, 4 had mild coagulatlon disorder with few treatment episodes. possible precipitating factors were coinfection with hbv, high alcohol consumation and first exposure to hcv contaminated blood products in an advanced age, but not intensive replacement therapy. very similar results for f vlll and vwf. since the factor viii level is kept steady above the level where there is an increased risk of haemorrhage, continuous infusion is haemostatically safer and more efficacious than bolus injections, another advantage is a progressive decrease of clearence during the first days after surgery which leads to a substantial reduction of factor concentrate consumption by avoiding the innecessary peaks of bolus injections. 22 children with severe form of haemophilia a undergoing elective surgery received continuous infusions with different plasma-derlved and recombinant f viii concentrates. before surgery, patients got bolus injections to raise the factor viii levels to more than 80 %. during continuous infusion factor viii levels were measured two to three times a day and the infusion rate of 4 to 5 iu/kg/h could be reduced on the second or third day to 2-3 iu/kg/h. the clinical efficacy was excellent with no bleeding events. in 5 children with vwd also undergoing elective surgery continuous infusions with humate pr were performed in the same way. no bleeding events were observed in these patients. none of the patients developed postoperative wound infections. the overall doses of f vtll concentrate 'were about 20-30 % lower than those required during replacement therapy with bolus doses. lg8 factor x frankfurt i : molecular and functional characterisation of a hereditary factor x defect (gla +25 lys) huhmann i., holler b., krinninger b., turecek p.l, richter g., scharrer i., forberg e., watzke h. univ. klinik f0r inhere med.i, abteilung for h~tmatologie und h~mostaseologie, w~en; immuno-ag, wien ; klinikum der j.w. goethe-univ. frankfurt am main, abt. f. angiologie. factor x (fx) is a vitamin k-dependent plasma protein which is activated either by fvila/tissue factor or ixaniila. fxa is the main enzyme for conversion of prothrombin to thrombin. the congenital fx-deficiency (stuart -prower-defect) being inherited as an autosomal recessive trait subsequently leads to bleeding diasthesis of varying severity. our propositus is a 24 year old patient presenting a mild bleeding tendency. his p'fi (36 sec) is within the normal range, the pt ( 73% of normal) is slightly reduced. the factor x antigen level is reduced to 55% of normal. molecular charactedsation of the genetic defect was performed by amplification of the eight exerts and exonintron junctions by pcr and subsequent direct sequencing of the products. in comparison to the normal sequence we could determine a single mismatch within exon ii resulting in the substitution of +25 gla (gaa) by lys (aaa). the mutation abolishes a naturally occuring mboll site in the dna sequence of exon ii. the status of the fx encoding alleles was determined in the propositus, his mother and one of his brothers by amplification of exon ii and restriction digest with mboll. these family members were heterozygous with respect to the mutation in exert ii. fx was isolated from plasma of the propositus by monoq ion exchange chromatography. performing clotting assays with purified fx frankfurt i we determined an activity of 89% of normal fx upon activation with rw, 77% upon intdnsic activation (aptt) and 81% upon extrinsic activation (pt). this compares well with the results obtained from the patient plasma ( pt 56%, ptt 55% and rw 57% of normal) when the reduced fx-ag-level of the plasma (55%) is taken into account. we therefore conclude that the substitution of gla +25 to lys results in a fx molecule which is severely defective in both the intrinsic and extrinsic pathway of blood coagulation. bleeding after cardiothoracic surgery is still a frequent, important and sometimes life-threatening complication. thus, the aim of this study was to examine routine parameters of hemostasis and their predictive values for severe bleedings. this prospective study included patients undergoing cardiopulmonary bypass surgery. blood samples were drawn preoperatively as well as 0, 6, 18 hours and 2, 3, 4, 5, 6, 7, 8 days after surgery. blood loss from drains, transfusion of blood products and other important clinical data were monitored apart from platelet count, hematocrit, thrombin time, thromboplastin time, aptt and levels of fibrinogen, atiii and c-reactive protein; soluble fibrin (sf) was measured via protamine sulfate aggregability and total fibrin(ogen) degradation products (ftdp) by an elisa from organon teknika. n= 109 patients were examined (age: 64__+9 y). they lost 750+__460 ml blood (mean+sd) into the drains within the first 18 hours after end of surgery. a severe bleeding was defined to exist, if the blood loss exceeded this range (> 1200 ml within 18 h). fibrin(ogen) split products proved to be a useful parameter in predicting the risk of severe bleedings : ftdp levels exceeding 12 mg/i at end of surgery (n = 105) had a negative predictive value of 94%, positive predictive value of 60%, specificity of 96% and a diagnostic efficacy of 91%. in contrast, soluble fibrin which correlated well with fibrinopeptidea (r>0.91, n= 20) did not correlate neither with degradation products nor with bleeding complications (n = 109). this observation does not match to the correspondence of sf with organ dysfunction during dic: sf reached a neg.predictive value near 95% and a diagnostic efficacy of >70% (pat. without antifibrinolytic drugs), which complies to findings from bredbacka (1994). other parameters were less predictive than ftdp and sf. therefore, further examinations are necessary to determine the value of soluble fibrin for a risk prediction of bleeding complications or dic. a differentiation of splits products deriving from either fibrinogen, fibrin or xl-fibrin will provide further insights into fibrin(ogen) metabolism. heparin induced thrombocytopenia represents a multicomponent syndrome associated with the use of heparin and related drugs resulting in not only thrombocytopenia, but also arterial thrombosis of varying magnitude. the initial diagnosis ofthis syndrome is usually made by clinical observation and a drop in platelet count. conventional diagnostic methods include platelet aggregation responses to patient's serum and ~4c serotonin release in response to patient's serum, aggregation/agglutination of patient's platdets in response to heparins and the detection of patients anti-heparin platelet factor 4 (hpf4-ab) ned-antibodies by using elisa methodology. several other individualized methods are also used to demonstrate platelet activation. to test the diagnostic validity of the platelet aggregation (pa) 14c serotonin release (sr) and the relevance of hpf~-ab 340 serum samples collected from patients with clinically eunfwmed eases of lilt syndrome were compared in parallel in various assay systems. the diagnostic efficacy of these tests varied from 60-74% with the pa test providing better results than others. when the pa test was compared with serotonin release, a poor correlation was noted (r=0.47). in contrast, the correlation between the pa and hp4-ab was somewhat better (r=0.66). in another study, blood samples collected from 50 patients treated with ahigh dose low molecular weight beparin for two weeks (80 mg o.d.) were tested. 20 of these patients showed a high titre of hpf4.ab without any decrease of platelet count. none of these patients were found to be positive in the 14c serotonin release assay. a third study included blood samples from dvt patients administered with iv heparin infusion, high dose sc lmw heparin (certoparin) and iv lmw heparin for the management of dvt. none of these patient groups (20-34) exhibited any hit responses, hmvever, the incidence of high hpf4-ab titre was found to be 53% in heparin, 36% in patients with lmw heparin iv and 26% in lmw heparin sc groups. pa and sr studies revealed 8% and 12% false positive ~ respeetively. these studies clearty suggest that the currently available ~ for laboratory diagnosis of hit syndrome are of limited value, and caution should be exercised in the interpretation of the results obtained with these tests. heparin-induced thrombocytopenia (hit) is one of the major severe side effects during treatment with heparin. in postoperative medicine clinical studies demonstrated the prevalence of hit with unfractionated over fractionated heparins. few data are available from the non-ope1"ative medicine and from patients without thmmboembolism before heparinization. in a controlled prospective randomized study the safety and efficacy of low-dose heparin was compared with a lowmolecular-weight (lmw) heparin over 10 days in bedridden medical inpatients (haemostasis, in press). 1968 patients were randomized and controlled for the development of thrombocytopenia. thrombocytopenia was defined as a platelet count below 40.000lid at day 10. 4 patients developed thrombocytopenia in the heparin group and no patient in the lmwheparin group (p<0.05). none of the patients with thrombocytopenia developed a thromboembolic complication. in a second prospective case control study 90 patients with side effects on anticoagulants were treated with lmw-heparin once daily subcutaneously for a period of 1 month to 9 years. platelet count was performed every 1 to 3 months. none of these patients developed thrombocytopenia during heparinization with lmw-heparin. it is concluded that hit is a very rare complication in nonoperated bedridden medical patients. a decrease of platetet count may occur in about 0.5% of patients receiving low-dose heparin. the incidence of hit with thrombosis during low-dose heparin and of hit during lmw-heparin in non-operated patients is manyfold lower and remains to be determined. terminology: instead of the term "hemorrhagic disease of the newborn (hdn)" the term vkdb should be used, since neonatal bleeding is often not due to vkdeficieacy and vkdb may occur after the neonatal period (i.e. after 4 weeks). definition: vkdb is a bleeding disorder caused by reduced activity of vkdependent coagulation factors which responds to vk. diagnnsis: in a bleeding infant a prolonged pt (inr > 3.5) together with normal fibrinogen and platelet count is almost diagnostic of vkdb. the diagnosis is proven, if vk shortens the pt (after only 30-60 minutes) and/or stops bleeding. classification: classification by age of onset into early (< 24 h~. classic fdav 2-7) and lale form (> i week <6 months), and by etiology into idionathic and ~ec0nd~'y. in secondary vkdb in addition to breast feeding other factors can be demonstrated, such as poor intake or absorption of vk and increased consumption of vk. vk-prophylaxis: benefits: oral and intramuscular (i.m) vk (one dose of i nag) prevents equally well the classic form of vkdb. lm. vk appears to be more effective in preventing the late form (times 15-> 50). the protection achieved by single oral prophylaxis (times 3-5) is improved by triple oral vk (times 15-30). risks: because of poten[ial ri~l~ associated with extremely high levels of vk and the possibility of injection injury, i.m. vk has been questioned as the prophylaxis of choice for normal neonates. since vk is involved not only in coagulation but 'also in carboxviation with multiple effects, excessive deviations from the low physiologic concentrations, which prevail in the fully breast-fed healthy mature infant should be avoided. proposal: repeated (daily or weekly) small oral doses of vk are closer to physiologic conditions than single i.m. bolus doses, which expose neonates to excessively high vk levels. the incidence of intracranial vkdb can be reduced if the grave significance of warning signs is recognized (i.e, icterns, failure to thrive, feeding problems, minor bleeding, disease with cholostasis). whether or not the more reliable absorption of the new mixed mieellar (mm~ nrenaral~i0n of vk can reduce the protective oral dose of vk-.prophylaxis has to be evaluated. before vitamin k(vk) prophylaxis was generally accepted in japan, the incidence of infantile vk deficiency was 1:4000 both idiopathic and secondary types. since 1981, 4 nationwide surveys have been conducted. the current incidence rate is now about one-tenth that in early 1980. however, in a small number of cases, vk deficiency occured despite prophylactic administration during the neonatal period. in order to clarify the absorption,excretion and transplacentel transport of vk in the perlnatal period,followlng studies were carried out. 1)hepaplastlntest(normotest) were performed on 65 women in the last stage of pregnancy and each coagulation factor was estimated as well. 2)correlatlons were made between mothers'and babies'hepaplastin test values. 3)transplacental transport of vk 2 was studied. the general activity of vk dependent factors in pregnant women was much higher than in non pregnant women. as far as the correlation between mothers'venous blood during delivery and cordvenous blood is concerned, in the group of mothers with hepaplastln test value of less than 120% of the normal adult value, the value of the bepaplastlntest was less than 30 % of normal adult value in the cord venous blood. we also demonstrated that vk passed through the placenta but only in small qualities. the point mutation g to a at nt 449 in exon v of the factor x gene (gin 102 to lys) has previously been found in two independent kindreds with fx deficiency. it occured in both families in an heterozygote state and was associated with two other genetic defect in the fx gene. we have identified another familiy in which this mutation occurs in a homozygote state. in this family the mutation is associated with the previously reported mutation gla 14 to lys which also occurs in a homozygote state. the pt and ptt of the proposita and her siter are markedly prolonged. the fx activity is reduced to <1% in the extrinsic system, to 30% in the intrinsic system and to 18 % after activation with rvv. the fx antigen is reduced to 20%. the coagulation profile of this family thus is identical with that of fx vorarlberg despite the fact that the fx vorarlberg kindred is only heterozygous for the mutation glal02 to lys. haplotype analysis could not rule out consanquinity with the fx vorarlberg kindred. these data suggest that the mutation at nt 449 which leads to a fairly dramatic amino acid change from glu to lys would indeed represent a polymorphism. to further address this question we cloned the fx gene in an expression vector (pcep 4) for transient expression in the human embryonic kidney cell line 293 and introduced the mutation at nt 449 by site directed mutagenesis. hereditary deficiency of factor ixa, a key enzyme in blood coagulation, causes hemophilia b, a severe x-chromosomelinked bleeding disorder; clinical studies have identified nearly 500 deleterious variants. the x-ray structure of porcine factor ixa shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for fx activation by phospholipid-bound flxa and cofactor villa. the 3.0 a resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (egf)-like modules; the n-terminal gla module is partially disordered. the catalytic module, with covalent inhibitor d-phe-pro-arg chloromethyl ketone, most closely resembles fxa but differs significantly at several positions. particularly noteworthy is the strained conformation of glu-388, a residue strictly conserved in known fixa sequences but conserved as gly among other trypsin-like serine proteinase. flexibility apparent in electron density together with modelling studies suggests that this may cause incomplete active site formation, even after zymogen activation, and hence the low catalytic activity of fixa. most hemophilic mutation sites of surface fix residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary flxa-fvilla-fx complex structure: the stabilizing fvilla interactions force the catalytic modules together, completing flxa active site formation and catalytic enhancement. factor x frankfurt i molecular and functional characterisation of a hereditary factor x defect (gla +25 ---, lys) huhmann i., holler b., krinninger b., turecek pi., richter g., scharrer i., forberg e., watzke h.. univ. klinik ftlr innere medi, abteilung for h~matoiogie und hamostaseologie, w~en; immuno-ag, wien ; klinikum der j.w. goethe-univ. frankfurt am main, abt. f. angiologie. factor x (fx) is a vitamin k-dependent plasma protein which is activated either by fvila/tissue factor or ixaniila. fxa is the main enzyme for conversion of prothrembin to thrombin. the congenital f×-deficiency (stuart -prower-defect) being inherited as an autosomal recessive trait subsequently leads to bleeding diasthesis of varying severity. our propositus is a 24 year old patient presenting a mild bleeding tendency. his ptt (36 sec) is within the normal range, the pt ( 73% of normal) is slightly reduced. the factor x antigen level is reduced to 55% of normal. molecular characterisauon of the genetic defect was performed by amplification of the eight exons and exonintron junctions by pcr and subsequent direct sequencing of the products. in comparison to the normal sequence we could determine a single mismatch within exon ii resulting in the substitution of +25 gla (gaa) by lys (aaa). the mutation abolishes a naturally occuring mboti site in the dna sequence of exon ii. the status of the fx encoding alleles was determined in the propositus, his mother and one of his brothers by amplification of exon ii and restriction digest with mboll. these family members were heterozygous with respect to the mutation in exon i1. fx was isolated from plasma of the propositus by monoq ion exchange chromatogrephy. performing clotting assays with purified fx frankfurt i we determined an activity of 89% of normal fx upon activation with rw, 77% upon intrinsic activation (aptt) and 81% upon extrinsic activation (pt). this compares well with the results obtained from the patient plasma ( pt 56%, ptt 55% and rw 57% of normal) when the reduced fx-ag-level of the plasma (55%) is taken into account_ we therefore conclude that the substitution of gla +25 to lys results in a fx molecule which is severely defective ip both the intrinsic and extrinsic pathway of blood coagulation. bleeding after cardioth~)racic surgery is still a frequent, important and sometimes life-threatening complication. thus, the aim of this study was to examine routine parameters of hemostasis and their predictive values for severe bleedings. this prospective study included patients undergoing cardlopulmonary bypass surgery. blood samples were drawn preoperatively as well as 0, 6, 18 hours and 2, 3, 4, 5, 6, 7, 8 days after surgery. blood loss from drains, transfusion of blood products and other important clinical data were monitored apart from platelet count, hematocrit, throm. bin time, thromboplastin time, aptt and levels of fibrinogen, atiii and c-reactive protein; soluble fibrin (sf) was measured via protamine sulfate aggregability and total fibrin(ogen) degradation products (ftdp) by an elisa from organon teknika. n= 109 patients were examined (age: 64+9 y). they lost 750+__460 ml blood (mean_+sd) into the drains within the first 18 hours after end of sur. gory. a severe bleeding was defined to exist, if the blood loss exceeded this range (> 1200 ml within 18 h). fibrin(ogen) split products proved to be a useful parameter in predicting the risk of severe bleedings : ftdp levels exceeding 12 mg/i at end of surgery (n = 105) had a negative predictive value of 94%, positive predictive value of 60%, specificily of 96% and a diagnostic efficacy of 91%. in contrast, soluble fibrin which correlated well with fibrinopeptide a (r>0.91, n= 20) did not correlate neither with degradation products nor with bleeding complications (n= 109). this observation does not match to the correspondence of sf with organ dysfunction during dic: sf reached a neg.predictive value near 95% and a diagnostic efficacy of >70% (pat. without antifibrinolytic drugs), which complies to findings from bredbacka (1994). other paramelers were less predictive than ftdp and sf. therefore, further examinations are necessary to determine the value of soluble fibdn for a risk prediction of bleeding complications or dic. a differentiation of splits products deriving from either fibrinogen, fibrin or xl-fibrln will provide further insighls into fibrin(ogen) metabolism. this study was conducted as a randomized parallel -group clinical trial comparing the safety and efficacy of a low molecular weight heparin {lmwh} -monoembolex sandoz and unfractionated standard heparin glfh) for the perioperative prevention of venous thromboembolie disease (dvt) following major surgms' in patients with gynecologic malignancy.. three hundred and twenty 4 women (six drop outsl werr randomized and received either 3 times daily 5000 [l" s.c. ul.'i-i (sandoz nuemberg germany] (n = 164) or once a day t5000 i~'v'. units s.c. monoembolex (n = 160) plus two placebo injections. heparin therapy was started the morning before opcrati(m and continued until the 7th postoperative day. up to the 10th poatop, day the incidence of dvt was 6.25 % (n = 10; incl. 7 pulmona~ embolisms pe) in the lmwh group and 6.10 % (n = 10; incl. 4 pe} in the ufh group. the overall incidence of clinically hemorrhagic wound complications was significantly decreased in the lmwh group 16.3 % (n = 2hi compared to the ufh group 26.8 % {n = 44; p < 0.0051. the incidence of major hemorrhagic episodes was 9.4 % in = 151 in the lmwh group and 14.0 %/n = 23) in the ufh group. this difference was not statisticauy significant. one case of fatal pe was observed in the lmwh -treated group. five women deaths in the lmwh group were observed during the study and 3 in the ufh group. this study demonstrates that the perioperative treaunent of low molecular weight heparins is more safety than standard heparins in gynecologic -oncologic patients undergoing major surge .ry. however, the incidence of thromboembohc complications is simmilar in both treatment regimes. to explore the effect of targeting an antithrombin to the surface of a thrombus, recombinant hirudin (hir) was covalently linked to the fab' fragment of fibrin-specific monoclonal antibody 59d8 (fab) resulting in a stable conjugate (hir-fab). in vitro, hir-fab was 9times more efficient than hir alone in inhibiting fibrin deposition on experimental clot surfaces in human or baboon plasma (p<0.01). to validate these results in vivo, hir-fab was compared to hir in a baboon model. the deposition of ill-in-labeled platelets onto a segment of dacron vascular graft present in an extracorporeal arteriovenous shunt was measured. blood flow rate was 40 ml/min. one hour local infusions of 4500 atu of either hir-fab or hir resulted in deposition of 0.16 x 109 and 2.17 x 109 plate!ets, respectively. equieffective dosages were 2000 atu hir-fab and 9000 atu hir resulting in deposition of 1.06 x 109 and 0.93 x 109 platelets, respectively. based on full dose response curves (n = 14), hir-fab was found to be > 4.5-fold more potent (based on activity) than hir. because of the small total amounts of antithrombins used and the short duration of these experiments, no significant systemic effects were observed. thus, fibrin-targeted recombinant hirudin prevents platelet deposition and thrombus formation more effectively than uncoupled hirudin in vitro and in an in vivo primate model. triabin, a 17 kda protein from the saliva of the assassin bug triatoma pallidipennis, is a new specific thrombin inhibitor (1). tt does not block the catalytic center but interferes with the anionbinding exosite of thrombin. the recombinant protein was produced with the baculovirus/insect cell system and used to study the inhibitory effect of triabin on thrombin-induced responses of human blood platelets and blood vessels. aggregation of platelets in tyrode's solution was measured turbidimetrically at 37°c. for the studies on blood vessels rings (2-3 mm) from small porcine pulmonary arteries were placed in organ baths for isometric tension recording. the integrity of the endothelium was assessed by the relaxant response to bradykinin. like hirudin, triabin inhibited the thrombin (0.1 u/ml)-induced aggregation of washed human platelets at nanomolar concentrations (ec50 = 2.6 nmol/l); whereas the adp-and collagen-induced aggregation were not suppressed. in pgf2c~-precontracted porcine pulmonary arteries, the thrombin (0.2 u/ml)-induced endothelium-dependent relaxation was inhibited by triabin in the same concentration range as found for inhibition of platelet aggregation. higher concentrations of triabin were required fo affect the contractile response of endothelium-denuded porcine pulmonary arteries to thrombin (1 u/ml). in all these assays, the inhibitory potency of triabin was dependent on the thrombin concentration used. these studies suggest that the new anion-binding exosite thrombin inhibitor triabin is one of the most potent inhibitors of the thrombin-mediated cellular effects. dept. of medicine, university hospital benjamin franklin, free university of berlin, dept. of medicine and dept. of surgery, heinrich-heine:university dusseldod after standardized training in home prothrombine estimation using the coaguchek system, 150 consecutive patients (p) who had st. jude medical aortic or mitral valve implantation were allocated to two random arms; 75 p were asked to control the inr themselves every third day. in the remaining 75 p anticoagulation was managed by the home physician without recommending an interval for these controls. all 150 p were monitored during the education period to a target therapeutic range of inr 3.5-4.0. p were asked to contact their home physician immediately if the inr was measured 0.5 below or above the target range (inr-corrider 3.0-4.5). all p had out-patient re-examinations every three months. thrombotic, thromboembelic and hemorrhagic complications were documented by the p using special documentation cards. the following findings were documented during the follow-up period: 4.49 0.9 0 the results of this randomized study demonstrate a significant improvement in the management of oral anticeagulation by home prothrombine estimation. significant (p<0.001) more inr measurements were found inside the target therapeutic range. moreover. bleeding and thromboembolic complications could be reduced (p = 0.038) in the study group with home prothrombine estimation. life-threatening thromboembolic and hemorrhagic complications were not observed in p who were on home prothrembine estimation, while three such events (2.72 %/year) were documented in group a. local vascular injury following ptca exposes circulating platelets to prodmmbogenic stimuli. by binding to platelet gp iiblliia fibrinogen crosslinks platelets, which represents the final common pathway of platelet aggregation. fradafiban (bibu52zw) is a non-peptide compound with effective, reversible inhibitory effects on fibrinogen binding to gp iib/ii/a on human platelets. in the first double-blinded, prospective phase ii study three escalating doses of bibu52zw as a continuous 24 h-i.v, infusion were tested in comparison to placebo in 65 patients with stable angina pectoris undergoing elective ptca. the mean receptor occupancy with 20rag, 40ms and 60ms per hour were 71.974, 84.5 % and 87.9% at 24 hours, respectively. as compared to placebo breeding time was significantly prolonged (7 vs 20rain) during fi-adaiiban infusion with a weak dose-dependency. platelet aggregation in platetet rich plasma ex vivo with collagen (2.0 and 4.0 gg/ml), adp (2.5 and 5.0 gmol/ml) or ca-ionophor a 23187 (2.5 and 5.0gg/ml) was significantly and dose-dependently inhibited as compared to placebo. using the two upper doses of fradafiban, we observed major bleeding complications in 8 patients requiring blood transfusions or vascular surreal repair. in these patients, too, maximal antiplatelet effects could be documented. these data sugest that bibu52zw is an effective fibrinogen receptor antagonist in patients. the requirement of ad hoc receptor occupancy determination or platelet function monitoring for safe and effective clinical use should be evaluated. in a placebo controlled interaction study 9 healthy volunteers were randomized to receive either a 24 hour infusion of peg-hirudin ( 0.02 mg/kg/h) after an i.v, bolus of 0.2 mg/kg + placebo, or 325 mg/day acetylsalicylic acid (asa) for three days followed by a placebo infusion or the peg-hirudin infusion + asa. each volunteer received all three treaments. there was a washout period of at least 14 days between the infusions. at short intervals aptt, activated clotting time (act), ecadntime (ect), alia-activity using the chromogenic substrate 2238, collagen-induced aggregation, platelet adhesion and platelet induced thrombin gene,ration time (pitt) were measured, bleeding time (simplate) was studied before drug administration, on day three before the infusion and 6 hours after start of the infusion.the infusion of peg-hirudin after 4 and 8 hours led to a mean hirudin plasma level of 1.8 pg/ml. asa markedly inhibited collagen induced aggregation as expected. the mean bleeding time was prolonged under the influence of peg-hirudin from 5.2 to 6.22 min, after asa from 5.8 -18.2 min and after the combination of peg-hirudin + asa from 5.4 -33.7 min. in each volunteer the bleeding time was longer under the combination than after asa alone. in two volunteers receiving peg-hirudin + asa the bleeding time measurement was stopped after 60 rain. none of the coagulation parameters or platelet function tests correlated with the prolongation of the bleeding time. however the bleeding time was excessively prolonged in those volunteers who had a marked prolongation under asa alone.the combination of hirudin at a higher dosage with asa probably is associated with a relative high risk of bleeding. either the hirudin dosage should be reduced if the combination seems feasabie or asa should be given after the end of hirudin treatment. fibrinogen with the sta/stago and the mla/dade systems correlated well, but neither system correlated well with the acl/il system. at iii, protein c, protein s, and anti-xa heparin assays using stago reagents performed as expected for normals and low abnormals on the sta. factor levels on the sta/stago system were less sensitive than factor levels obtained with the dade reagents on the mla or fibrometer. using the sta/stago system, thrombin time results correlated well with the aptt and heparin levels. the thrombin time was not associated with additional manipulation for assay preparation, nor any cross-contamination of reagent or sample, since on the sta reagents do not come into contact with tubing. the sta was not sensitive to hemolytic, icteric or lipernic samples for clotting assays artd showed the same sensitivity as the mla for chromogenic assays. the overall data comparisons, high throughput, minimal operator intervention for reagent/assay change and ease of operation warrant further evaluation of the sta hemostasis analyzer. a. wehmeier, d. s6hngen, c. rieth klinik for h#,matologie, onkologie und klinische immunologie der heinrich-heine-universit~it d0sseldorf hirudin selectively inhibits thrombin by direct interaction. because the effect of hirudin is independent of antithrombin iii and other factors, it seems an attractive alternative to current anticoagulants. however, it is uncertain whether hirudin influences plateletassociated thrombotic disorders and how it compares with conventional and lmw heparin. we investigated the effect of 2 recombinant hirudin preparations (rhein biotech, dt3sseldorf) on platelet function tests: in vitro bleeding time, adhesion to glass beads, aggregation in platelet-rich plasma and whole blood. hirudin was used in concentrations of 0.1-100 i.tg/mi, and was compared to trisodium citrate (0.38%), conventional heparin (50 iu/ml) or lmw heparin (fraxiparin, 500 iu/ml). both recombinant hirudins showed normal activity in thrombin neutralization tests, and prolongation of thrombin time and aptt. however, in vitro bleeding time was not prolonged by hirudin, but was more than doubled by addition of conventional and lmw heparins. platelet retention to glass bead columns was reduced by hirudin in a dose-dependent manner to about 40% but was more effectively reduced by both heparin preparations and citrate. hirudin had an inhibitory effect on p!atelet aggregation in prp induced by thrombin, collagen, and predominantly epinephrine but not adp and ristocetin. in whole blood, a small effect could only be observed with hirudin concentrations of >25 ~g/ml as compared to citrateanticoagulated blood. in summary, thrombin inhibition by recombinant hirudin has little effect on in vitro platelet function tests in comparison to heparins and calcium depletion. the role of endothelin (et), prostaglandins and the coagulation system in the pathogenesis of acute renal failure is still to be defined. in anaesthesized pigs the effects of i.v. infusion of et (3 /~g/kg) alone (group 1, n=6) and after pretreatment with the potent thrombin-inhibitor hirudin (0,5 mg/kg)(group 2, n=6) on haemodynamics, coagulation parameters (factor viii, antithrombin iii, precallicrein, fibrin monomers, aptt) and prostaglandins were investigated. plasma renin activity (pra)-, creatinine clearance-, urine volume-measurement and blood gas analysis were performed hourly. et-infusion caused an initial bp-reduction and marked hr-reduction followed by a transient bp-elevation and hr-reduction. activation of platelets can be directly measured by flow cytometry using monoclunal antibodies. in an in vitro study the effect of the thrombin inkibitors argatroban, efegatran, dup 714, recombinant hirudin and peghirudin on platelet activation induced by various agonists was studied in whole blood. blood was drawn from normal human volunteers using the double syringe technique without use of a tourniquet to avoid autoaggregatiun of platelets. for anticoagulation of blood the thrombin inhibitors mentioned above were used at a final concentration of 10 ~tg/ml each. blood samples were then incubated at 37°c either with saline, r-tissue factor (rtf), arachidonic acid (aa), adenosine diphosphate (adp) or collagen. at definite times (1, 2.5, 5, 10 rain) aliquots were taken and after various steps of fixative procedure the percentage of platelet activation was measured by means of fluorescent monoclonal antibodies to platelet surface receptors gpiiia (cd-61) and p-selectin (cd-62). the agunists used induced a platelet activation of 37.4 + 15.2 % (rtf), 65.1 + 12.1% (aa), 19.3 + 7.4 % (adp) and 27.1 + 12.8 % (collagen). flow cytometric analysis showed that all thrombin inlaibitors studied caused a nearly complete inhibition of r-tissue factor-mediated platelet activation. in contrast, after induction of platelet activation with the other agonists an increased percent cd-62 expression was found showing a strong platelet activation with a maximum at the same times as in non-anticoagulated blood. in conclusion, the results show that in whole blood thrombin inhibitors are effective in preventing platelet activation induced by r-tissue factor. the formation of active serine proteases including thrombin may be effectively inldbked by these agents. the observations further suggest that, while thrombin inkibitors may control serine proteases, these agents do not inhibit the activation ofplatelets mediated by other agonists. this work was supported by the grant bmft 07nbl01. animal experimental studies on the pharmacokinetics of peg-hirudin e. bucha, a. kossmehl, g. nowak max-pianck-gesellschaft e. v., arbeitsgruppe "pharmakologische h~imostaseologie", jena hirudin, when complexed with polyethylene glycol (peg), increases its molecular weight from 7 to 17 kda, thereby preventing extravasation of this drug. peg-hirudin is distributed almost only in the intravascular blood space. in addition, its increased molecular weight retards the renal elimination. the elimination half-life of hirudin in rats (58 + 12 min, as determined) is increased five-fold (244 ± 18 min). with the same hirudin dose applied, the blood level of hirudin is increased 19-fold, measured in the 13-elimination phase. in the urine of rats, 2 -3% of the hirudin activity were recovered following hirudin administration, but 48% could be detected after peg-hirudin had been applied. after subcutaneous administration of peg-hirudin, the trnaxwalue is reached at 380 rain (r-hirudin: 65 min); the cmax-value is increased 3-fold, compared to that of r-hirudin (2.5 pg/ml). 24 hours later, still one fifth of the maximum concentration (cma,) is present in the blood, and the renal elimination is still retarded. in the urine of rats, 12% of the hirudin activity applied were recovered in the 24-h urine sample. with intact renal function, following subcutaneous administration, peghirudin is abte to produce a constant blood level of hirudin over a long pedod. thrombin inkibitors such as r-hirudin (rh), argatroban (a), efegatran (e), and peghiradin (ph) are currently undergoing extensive clinical trials in such cardiovascular indications as ptca, ami, and treatment of unstable angina. a rapid assessment of the anticoagulant actions of these agents is, therefore, crucial to assure their efficacy and safety. currently, act and aptt are used to measure the anticoagulant effect of these agents. we have utilized a dry reagent technology based on the motion of paramagnetic iron oxide particles (plop) to measure the antithrombin effects of various thrombin inhibitors (cv diagnostics, raleight, nc). the heparin monitoring card has been modified to measure antithrombin agents in various anticoagulant ranges for (a)0 (e), (rh), and (ph). blood samples drawn from patients treated with (a) and (rh) have been evaluated and concentrations of these agents have been calculated using an external calibration curve. in the in vitro setting, citrated whole blood or citrated frozen plasma can be used to evaluate the anticoagulant effects of these agents. the results obtained are comparable to the act which is conventionally used for the monitoring of these agents. both (rh) and ( period. we would like to present a case of heparin-induced-thrombocytopenia (hit) in a 47 years old woman who underwend open heart surgery. she suffered from a combined aortic valve disease and leading stenosis. laboratory analysis showed constant low platelet counts (50/nl) without heparin application, so that an idiopathic thrombocytopenlc purpura was suspected. but platelets also decreased after heparin application. heparin-antibodies were found in the heparin induced platelet activation assay (hipaa). treatment with corticosteroids and immunoglobulines, respectively, showed no improvement but the patient unfortunately developed a pneumonia with legionetla pneumophila. therefore, the only suitable anticoagulant for the necessary aortic valve replacement was hirudin: a bolus injection of r-hirudin of 0,75 mg/kg b.w. was administered 10 min. bevore start of the extracorporal circulation (ecc), the heart-lung machine (hlm) was primed with 6 mg r-hirudin and another bolus of 5 mg of r-hirudin was administered. additionally 10 mg of r-hirudin was applicated to the cell-saver-reservoir. during the period of ecc ecarin clotting time and aptt values were taken every ten minutes for monitoring r-hirudin concentration. the postoperative anticoagulation was performed with a constant infusion of r-hirudin starting eight hours after the end of ecc and monitored by aptt. due to mechanical aortic valve the further anticoagulation was performed with phenprocoumon, starting 3 days postop. the therapy with hirudin showed no side-effects. hirudin, threrefore seems to be a suitable anticoagulant in patients with high risk for bleeding complications like this. doses fi:om 2-30 mg/kg gave similar post-op blood loss measurements without s dnseresponse (4-15 oc/kg) (less blood oozing than a historical heparin control but equivalent post-op blood loss; 10 q-3 ec/kg). doses >30 mg/kg showed more intra-op blood loss than the lowe~ doses, but equal post-.op blood loss. the bleeding time test was less elevated than for heparin. platelet counts and hematoerit did not vary except for hemodihition on pump. liver enzymes did not vary significantly pre-op to post. act values showed arg was eliminated (dose-dependently) by 1 hour post-op. dogs were hemodymamieally stable during the peri-operative period, and overall gave predictable responses to arg (as opposed to variable responses to heparin). in a substudy it was demonstrated that hypothermia did not affect the activity of arg, nor did varioos formnlations. this dose finding study strongly suggests that arg may be a safe and effective alternative to heparin for patients undergoing cpb. this is particularly important for the growing population of patients with hit who require cardiac surgery, for which no anticoagulant alternative is presently available. three recent clinical tdals with r-hirudin (timi 9, gusto 2 and hit) have shown that the risk of severe haemorrhagic side effects was strongly associated with high aptt-levels. the large intedndividual variability of the aptt and the lack of a linear dose-effect ratio, however, limits its value for reliable monitoring of the anticoagulant effect of hirudin since even severe overdosage due to impaired renal elimination may not be detected with this assay. we have therefore evaluated the ecadn clotting time (ect) as descdbed by nowak and bucha (thromb. haemost. 1993; 69: 1306) under conditions which allow conclusions on its reliability in the clinical situation.for this, citrated venous blood obtained from healthy volunteers, patients with unstable angina pectoris, and patients treated with marcumar was supplemented with different concentrations of peghirudin. measurements of aptt and ect were made in duplicate. in contrast to the aptt, the ect showed a close, linear relationship with peg-hirudin plasma concentrations in the range of 100 and 10 000 ng/ml. the lineadty of this relationship was not affected by the presence of unfractionated or low molecular weight hepadns in concentrations of up to 10 pg/ml. the ect was not affected by fibdnogen concentrations 60% below normal. a somewhat higher slope but no change in linearity was found in plasma from marcumar-patients with quick-values between 20 and 32%. no significant differences were found between values measured in citrated blood or plasma or using different coagulation timers. the most potent thrombin inhibitor containing a benzamidine moiety is napap (k i = 6 nmol/i). unfortunately, the pharmacokinetic properties (fast elimination by hepatic uptake and biliary excretion, poor enteral absorption) are unsuitable for the use of napap as an oral anticoagulant. the application of choice of a synthetic thrombin inhibitor would be the oral one, therefore, we looked for other lead structures. with the nc~-arylsulfonylated piperazides of 3-amidinophenylalanine we found a new group of derivatives which inhibit thrombin with ki-values in the nanomolar range. the piperazides exert anticoagulant activities with high selectivity, leaving activated protein c and components of the fibdnolytic system unaffected. in rats, the piperazides are rapidly eliminated from the circulation (tl/2 ~ 10 min) upon i.v. administration, too. after oral administration, the systemic bioavailability is low. upon intraduodenal administration of high doses widely varying blood levels were seen, depending on the mode of administration. to cladfy the importance of a possible hepatic first pass effect we studied in more detail the pharmacokinetics of the n~-(2naphthylsulfonyl)-3-amidinophenylalanine n'-acetylpiperazide in rats using hplc-analysis. like other benzamidines the piperazide is excreted via the bile to a high extent. enteral absorption rates of about 20 % are found after blocking the hepatic uptake and biliary excretion. hence, a hepatic first pass effect appears to be the main reason for low systemic bioavailability after orallenteral administration. at the same time, fast elimination from the circulation by hepatic uptake is the main problem for maintaining effective blood levels with benzamidines. therefore, the elucidation of the structural elements influencing the absorption and elimination processes of these types of inhibitors is necessary. the piperazides of 3-amidinophenylalanine bear the possibility to easily introduce a wide variety of substituents on the second nitrogen of the piperazine moiety. a 69-year-old female patient with diabetic nephropathy increasingly developed signs of allergisation combined with dyspnea, erythema, pruritus, and circulatory insufficiency two months after start of heparin-anticoagulated haemodialysis und initial surgical application of a double lumen venous catheter. in addition, growing thrombocytopenia was observed involving a drop in platelets by 50%, compared to the initial values. the haemodialytic efficiency was reduced by massive thrombosis of the dialyzer and subsequent repeated interruption of treatment. at the end of may 1995 heparin antibodies were detected and the hat diagnosis was confirmed. immediately afterwards, haemodialysis treatment was continued, applying hirudin as anticoagulant. using steam-stedlised haemophan dialyzers and 0.14 mg/kg r-hirudin (iketon, italy), the minimum therapeutic blood level of hirudin (0.4 pg/ml whole blood) was reached. this provided therapeutically relevant blood level conditions during a 4.5h haemodialysis. more than 80 regular haemodialyses were run without problems. in all hirudin-anticoagulated haemodialysis treatments the ecarin clotting time was used as the method of choice for bedside blood level and dosage control. after the 34th haemodialysis, the frequency was reduced from 3(4) to 2 haemodialyses a week. accordingly, the hirudin dose was increased to 0.2 mg/kg. the creatinine clearance increased continuously from initially 2.6 to 10.4 ml/min after the 13th week of hirudin-anticoagulated haemodialysis. platelet count and haemodialytic efficiency normalized. we could demonstrate that the regular use of hirudin as anticoagulant along with dialyzers impermeable to hirudin enables very good results in haemodialysis treatment in heparin-associated thrombocytopenia, hirudin is suited for use as anticoagulant in problem patients with hepadn-induced allergy when combined with a drug monitoring method fit for bedside use. capillary electrophoresis methods provide a fast measurement of proteins. thus we developed for pharmacokinetic measurements of r-hirudin and peg-hirudin capillary electrophoresis methods. for the measurement of r-hirudin we used fused silica capillary and a borate buffer. this buffer was used to detect r-hirudin, but could not be used to measure peg-hirudin. for simultaneous measurement we used a neutral capillary to prevent protein absorption to the capillary wall. the buffer was a 20 mm tricine buffer (ph = 8.0 field strength 500 v/cm). it resolved r-hirudin from peg-hirudin at 214 nm using reverse polarity. a linear correlation between the peak area and the concentration was found between 80 pgtml and 10 mg/ml for hirudin (r 2 = 0.99) and between 2,5 and 10 mg/ml for peg-hirudin (r2= 0.99) was found by coinspiking of human plasma and urine with r-hirudin and peghirudin the two proteins were completely resolved. a linear correlation between the peak area and the concentration was found. the method separates r-hirudin from peg-hirudin and may be applied to biological systems to measure the concentration of r-hirudin. triabin is a thrombin inhibitor from the saliva oft. pallidipennis structurally unrelated to any protease inhibitor known and which probably functions by an interaction with the anionbinding exosite of thrombin. we used sf9 insect cells infected with recombinant baculovirus to produce sufficient triabin for a detailed biochemical characterization. the activity of the protein purified from cell lysates was assessed in a fibrinogen clotting assay and was found to be similar to that of the natural protein. a 4-fold prolongation of thrombin-clotting time and aptt was achieved with 22 nm and 600 nm triabin, respectively. a kinetic analysis of the thrombin-catalyzed fibrinopeptide a release from fibrinogen showed that triabin is a tight-binding inhibitor. using the graphical method of dixon, the ki was determined to be 3 pm. introduction: thrombocytopenia is a common adverse effect of heparin therapy, in type ii hit platelet decrease induces severe complications. we here present two special cases of type ii hit. case report i: a 66 year old male patient with dvt of the left leg was treated with therapeutic doses of heparin. from the first to the 12th day of therapy, platelet count decreased from 122000 to 54000/ui. hit was confirmed by hipa-test, heparin therapy was s~opped and treatment with the heparinoid orgaran n was started. during the following days, arterial thromboses in the right a. femoralis occurred. several thrombe~tomies were not successful and although orgaran ~" was stopped because of suspected crossreactivity, amputation of the right leg could not be avoided. during the following days under hirudin-treatment platelet count normalized and no further complications occurred. case report 2: a 74 year old female patients suffering from hip fracture was treated by surgery with tep-operation and received prophylactic heparin treatment. after 6 days, platelet count decreased from initially 170000 to 8000/ul and dvt of the right leg was diagnosed. on the same day, severe bleeding into the left leg was observed and hemoglobin concentration was diminished to 7.8 g% (before surgery 16.0 g%). hit was confirmed by hipa te~t, heparin was stopped and treatment with orgaran started. thrombocyte count normalized and no further complications occured. conclusion: hit type ii can cause severe bleeding as well as thromboembolic complications. because of possible cross-reactivity between heparin and orgaran~,, hirudin should be given in hit patients. currently thrombin time (ti), aptt, activated clotting time (act) or anti ila -activity (alia), measured by a chromogenic substrate test are used to monitor hirudin treatment or prophylaxis. the "13" responds very sensitive to hirudin plasma levels end thus requires variable thrombin concentrations. aptt appears to be more adequate, however, it shows large interindividual variations and does not respond sensitive enough to higher hirudin concentrations. act is a simple whole blood clotting assay, but it is strongly influenced by the blood collection technique. the ecadn clotting time (ect) is a new clotting assay, recently described by nowak and bucha (thromb.haemost 1993, 69,1306) . it measures the clotting time of citrated blood or plasma after prothrombin activation by ecarin, a snake venom of echis carinatus. ec.t shows a linear dependence on different hirudin concentrations over a wide concentration range ( e.g. 0.1-5 pg/ml). in a clinical interaction study healthy volunteers were administered hirudin, asa or both. 15 male volunteers received an i.v. infusion of peg-hirudin (0.02 mg/kg/h) for 24 hours after an initial i.v. bolus of 0.2 mg/kg to compare the sensitivity and reliability of ect with aptt, l-r end act. the act was measured on the hemochron 801, usa, ect on a fibrin timer, aptf using the aptt lyophylized silica reagent by il and alia on an acl (il-milan) with the chromogenic substrate 2238. all tests were performed in duplicate. ect was more sensitive to different hirudin concentrations than aptt, or act. the ect results were better correlated with the alia-activity than ap'l"r and act. the lower detection range for ect is 0.05 pg/ml hirudin. ect is a very sensitive, simple and reliable test for the monitoring of hirudin treatment and prophylaxis. recombinant and synthetic inhibitors of thrombin such as hirudin, efegatran and argatroban are currently in various phases of clinical trials in several surgical and medical indications. the therapeutic effects of these agents are usually monitored by aptt whereas in cardiovascular indications, cefite act and hemotech® act are used. the reliability of both aptt and the act tests in predieting the safety of various thrombin inhibitors has been heavily debated. furthermore, some of these inkibiturs are administered simultaneously to heperinized or coumadinized patients and the obtained aptt and act results do not lady refleot the effects of these agents. fcafin is a snake venom derived fi'om echis carinatus which converts prothrombin into mesothrombin, targeting the arg~3-ile tm bond between the a and b chains of prothrombm. while thrombin inhibitors are capable of inhibiting mesothrombin, atiii/beparin complex does not have any effect. using purified ecarin, nowak and bucha (1993 thromb haemost 69:1306) proposed to assay hirudin. since thrombin inhibitors exhibit similar mechanisms of thrombin inhibition, ecarin clotting time (ect) was evaluated to test its diagnostic efficacy in various experimental and clinical settings. lyphilized eoarin was obtained from knoll ag, ludwigshafen, germany). concentration dependent clotting times for himdin, efegatran and argatroban were obtained in a range of 0-15 p.g/ml. all of the antithrombin agents produced a concentration dependent prolongation of ect and showed va~angpotendies inthe order ofefegatran> argatreban> hirudin, on a gravimetriebasis. on a molar basis, the anticoagulant order of potency was found to be hirudin> afegatran> argatroban. utilizing the ect, the effect of these inhibitors on patients undergoing bolus or infusion therapy, resulting in a concentration level of ~ 10 gt g/rnt, have been measured. unlike such global tests as pt and aptt, patients receiving simultaneous heparin or oral anticeagulants can be monitored for antithrombin specific prolongation ofthe ect. plasma samples from heparinized (aptt 45-90 sec) or coumadinized ('pt 15-25 see) patients, supplemented with argatroban or hiredin did not show any differences m the ect. a medified ecarm act comparable to the celite act has also been developed. initial results demonstrate that this test is not affected by aprotinin, heparin and reduction of the prothrorabin complexes in the inr range of 1.5 -3.0. these results indicate that ecarin based clotting times provide slx~etlie ~lts of circulating levels of thrombin inhibitors, which can provide reliable information to optimize their safe(y and efficacy. r-hirudin is a highly potent and selective inhibitor of the serine proteinase thrombin. after intravenous administration, r-hirudin is eliminated exclusively with the urine. its plasma half-life is very short, 1-2h. peg-hirudin is a derivative produced by coupling polyethylene glycol (peg) to a specially designed recombinant hirudin mutein. peg-coupling results in a considerable prolongation of the plasma half-life of peg-hirudin, compared to r-hirudin. after intravenous administration of r-hirudin into rats, a very small amount of ,,hirudin-like" activity (2 -4% of applied activity) was recovered in the urine. in contrast, after peg-hirudin had been administered, more than 30% of the applied activity could be recovered in rat urine. these results suggest differences in the renal metabolism of peg-hirudin and r-hirudin. within the scope of pharmacokinetie studies in rats we investigated the appearance of biologically active metabolites of peg-hirudin after kidney passage in urine. affinity chromatography on immobilised thrombin was used as a quick and gentle method in searching for biologically active hirudin metabolites in rat urine. but it had to be completed by anion-exchange and/or reversed-phase chromatography to ensure that all active metabolites were detected. the isolated biologically active metabolites were purified by reversed-phase hplc and were biochemieally characterized. in previously reported studies we found a hlrud n derivative consisting of the amino acids 1-50 as the main metabolite in rat urine following intravenous administration of r-hirudin. this metabolite was not detected in the urine after administration of peg-hirudin, confirming the suggestion of a different renal metabolism. carrageenans are high molecular weight sulfated polygalactans of plant origin (derived from red algae) with anticoagulant properties. in previous studies we investigated the anticoagulant activity of lambda-carrageenan, a highly sulfated type of carrageonans. unlike heparin, lambda-earrageenan exerts its anticoagulant activity primarily through direct inhibition of the serine proteinase thrombin. only a part of its antithrombin activity is indirectly mediated through antithrombin iii. to investigate relations between molecular weight and biological activities, tambda-carrageenan has been hydrolysed and fractionated. the molecular weight has been determined with the aid of size exclusion hplc using dextrans as molecular weight standards. the degree of sulfation has been determined by anion-exchange hplc. we have obtained low molecular weight lambdaearrageenans ranging from 10,000 dalton to 100,000 dalton with degrees of sulfation of 13 -17% and 33 -38%. the anticoagulant and antithrombin activity of low molecular weight carrageenans have been determined using coagulation assays and purified systems, and we have compared their activities with those of heparin and other sulfated polysaecharides. further, we have investigated the ability of lambda-carrageenan and its low molecular weight derivatives to inhibit the activity of human blood phagocytes. the activity has been determined by measuring the cellular chemiluminescence in a mieroplate himinometer using a himinol-dependent assay and zymosan as phagocytosis activating agent. we have used an assay in human whole blood and assays with isolated human mononuclear and polymorphnuclear cells. the anticoagulant activity and also the ability of carrageenans to inhibit the activity of human macrophages decrease with decreasing molecular weight and decreasing degree of sulfation. the natural ocouring, yellow pigment curcumin is the major component of tumeric and is commonly used as a spice and food-coloring agent. since curcumin has been reported to have anti-tumorpromoting, antithrombotic and anti-inflammatory properties, we studied, whether curcumin acts on the transcription factors ap-l(jun/fos) and nf-~:b in cultured endothelial cells (ec). when ec were cultured in the presence of curcumin, electrophoretic mobility shift assays (emsa) demonstrated, that binding of endogenous ap-1 to its dna recognition motif was suppressed. inhibition was due to direct interactions of curcumin with the dna-binding motif for ap-i. enhanced ap-1 binding, induced after tnfa stimulation of ec, was decreased in cells pretreated with curcumin. this resulted in reduced transcription and expression of tissue factor, known to be controlled by ap-f and nf-~b. nuclear run on assays proofed, that curcumin directly reduced the tnfa mediated transcription of genes, regulated by ap-1, as tf, endothelin-1 and c-jun. thus, curcumin did not only suppress apl(jun/fos)-binding, but also inhibited tnfa induced jun transcription, transient transfections with tissue factor promotor plasmids confirmed, that inhibition by curcumin was dependent on intact ap-i sites. beside its effect on ap-l-binding, curcumin reduced the radical dependent activation of nf-kb due to its antioxidant properties, however, this inhibition was indirect and less prominent. the relevance of the in vitro data was confirmed in vivo in mice bearing meth-a-sarcoma. when mice received curcumin before tnfa was injected, tumors showed reduced ap-1 activation. simultanously fibrin/fibrinogen deposition decreased, most probably due to reduced tissue factor expression. thus, curcumin inhibits ap-t activation and expression of endothelial genes controlled by ap-t in vitro and in vivo. (jung, 1991) . additionally, haemorhenlogical parameters (plasma viscosity, erythrocyte aggregation) were measured. in all patients aptt, bleeding time, platelet adhesiveness, von wiuebrand f~ctor and factor viii concentration and activity were determined. the patients with von willebrand disease showed characteristic morphological changes of capillary geometry. tortuosity of nailfold capillaries was markedly increased as well as the diameter of capillariez on the arterial and venous side. plasma viscosity was significantly low. multiple parameter analysis concerning to galen and gambino (1983) and using the parameters ,,plasma viscosity below 1.25 mpas", ,,torquation index higher than 5", ,,erythrocyte column diameter bigger than 16,9 gin" showed a positive predictive value of 100 %. capillary diameter and capillary tortuosity have a positive predictive value of 91,2 %. additionally, a reduction of the vasomotorie reserve and/or a decreased erythrocyte velocity in the capillaries below the reference range was found in most of the yon willebrand patients. it was quite remarkable, that 16 of 40 of the yon willebrand patients showed significant capillary bleedings. these findings confirm some former observations (e.g. o'brian 1950) and preliminary reports of our group (koscielny 1994). polymerase chain reaction (pcr)-based quantitation of mrna transcripts is an important tool in the investigation of the underlying molecular defects in inherited platelet disorders, such as the bernard-soulier syndrome. however, for the exact quantitation of mrna a number of methological requirements has to be met. first, a standard (s) mrna must be synthesized which is able to undergo the same processing as the target wild type (wt) mrna. secondly, the quantitation step following the pcr must differentially recognize standard and target dna, and thirdly, the assay must be precise with respect to both inter-and intraassay variability. in order to satisfy these requirements we constructed a s-gpib mrna which is identical to the wt-gpib mrna except a 13 bp long primer recognition site at its 5" end allowing differentiation between the pcr amplified wt-or s-gpib cdna through incorporation of a fluorescein or biotin labelled 5" primer. both standard and w[ gpib mrna showed identical amplification kinetics in the pcr reaction. the amplified dna was quantified using an dna binding assay. in this assay binding of amplified dna to gcn4 fusionprotein-coated microtiterplates is measured. since the gcn4 binding motif is incorporated into the wt-and s-gpib cdna through an identical 3" primer, competition between s-and wt-cdna during amplification has been analyzed. at a given concentration of 250 nm of gcn4. primer no competition between the sdna and wt-dna for the primer was observed during 25 pcr cycles. the sensitivity limit of the assay performed in this way was 250 amol wt-gpib~, dna, and intraassay variability reached from 1.66% to 6.72% calculated for 100 fmol and 5 fmol dna, respectively. to sum up, combination of rt-pcr with the amplified dna binding assay and usage of an internal standard mrna allows sensitive and accurate quantitation of gpiba mrna in human platelets. since upa and thrombin are main conrtibutors to the process of proliferation and migration of vascular smooth muscle cells (vsmc), which is part of the pathogenesis of atherosclerosis. we are currently assessing the role of spatial expression of upa and thrombin receptor (tr) on cells with human carotid artery plaques (n=10). we have used a double immunolabeling approach, combining anti-upa and anfi-tr antibodies. to identify the different cell types, we used the following antibodies: anti a-smooth muscle actin (a-sma) for smooth muscle cells, ulex europaeus agglutinin i (uea i) for endothelial cells, inflammation cell cocktail (cd68+cd45) for monocyte/macrophage and lymphocytes and an anti-proliferation cell nuclear antigen antibody (pcna) to stain proliferating cells. in the carotid atherosclerotic plaques, upa immunostaining was distributed focally, preferentially in the fibrous cap and some cells of the foam cell rich region (fcrr). it was present in distinct patterns: cytoplasmic staining. tr staining was distributed similar to upa staining. with double staining combining anti upa antibodies with anti-tr antibodies, cellular co-localisation of both upa and tr was demonstrated. these cells were identified as smooth muscle cells by -sma. inflammatory cells were mainly localized within the fcrr, they only stained for upa. in conclusion: our data demonstrates that upa and tr are coexpressed in vsmcs in human carotid artery atherosclerotic plaque tissue. we therefore conclude, that the mitogenic activity of upa is associated with the thrombin signalling pathway. in the proficiency test of the ,,deutsche gesellschaft flir klinische chemie" (dgkc) 1/95, 5 lyophilised plasma samples (immuno ag) were sent to participants: a normal plasma and 4 plasmas from persons under oral anticoagulation (oac-plasmas. inr 1.9 to 3.7). the participants (n=552) returned the pt times obtained and in most cases (n=355) also the isi value for the thromboplastin used (isi of pack insert). the inr was calculated using the pt of normal plasma and the isi of pack insert (method i). two additional methods for inr calculation were compared with method i. according to the concept of calibrated plasmas (houbouyan et al., t993), a calibration curve was constructed using the normal plasma and the 30ac-plasmas. the inr calculated using the pt •fn•rma¿ plasma and the laboratory-specific isi value given as 1/slope of the calibration curve (method ii) or was read off directly (method hi). for inr values, calculated by the 3 methods from the participants data (n=355), outlier elimination (2sd, iterative) was performed. the inr mean values for all 3 calculation models remain in a narrow range. using calibrated plasmas (method 1i and m), less outlier were eliminated and cv's obtained were smaller than using the conventional procedure ( i ). obviously, the inr inherited problems, such as accurate isi value, pt value of normal plasma and instrument/laboratory influences on isi, can be reduced using calibrated oac-plasmas. practical approach and educational considera-tions of home prothrombin time estimation a. bernardo, a. bernardo, c. halhuber herz-kreislauf-klinik, bad berleburg, germany specific training is necessary for the patient to achieve reliable and reproducible results in prolhrombin time measurement. the training scheme is based in many respects on experience with similar training courses for home control and management of diabetes and asthma. the education program is divided into a theoretical and a practical part. the theory part has group sessions of twenty patients of a time. the practical course is reduced to a maximum of five patients. the sessions are conducted by a medical doctor and by specialized medicaf/technical assistants. on average eight hours of theoretical education and two hours of practical training are sufficient. the contents of the theoretical lessons are: • need for anticoagulation after heart valve replacement, • potential interaction between anticoagulants and other medication, • accurate recording of the measured prothrombin time results, • techniques of prospective determination of the necessary amount of anticoagulant, • calculation of the individual doses, • potential pitfalls and mistakes, • corrections in case of over-and under-dosage, • early recognition of thromboembelic and/or bleeding complications. an alternative is a full-day intensive course which can be held during the weekend. our recently reported (1) observation that oral anticoagulant treatment causes an increase of heparin cofactor ii (hc ii) activity in plasma is now confirmed by a more extensive study. in 43 thrombophilic patients who were on vitamin k antagonist therapy (marcumar r) we found a median hc ii level of 142 % as compared to 119 % for 72 thrombophilic patients without any therapy (p < 0.002" ) and 104 % for 59 healthy controls (p < 0.001" ). moreover we observed that the increase of hc ii level was significantly correlated with increasing inr-values (r = 0.63, p < 0.001). follow-up observations on some patients showed, however, clear differences in the levels of hc ii activity after onset of vitamin k antagonist therapy. thus, some patients responded rapidly with a significant increase in activity ("strong responders") while others showed only slight changes ("weak responders"). in conclusion, the determination of hc ii activity may result in an improved estimation of the risk of bleeding, especially in high intensity treated patients (inr > 3.5). after intracoronary stent implantation an aggressive oral anticoagulation (oac) therapy is mandatory. to find out whether coagulation activation occurs after coronary stent implantation during high dose oac therapy markers of plasmatic coagulation and d-dimer were measured. patients 5 male patients (average age 57 years) were examined. blood samples were taken before and right after stent implantation and during the following week. patients got 30 mg phenprocoumon during the first three days and additionally heparin and acetylsalicylic acid (asa) were given. methods ptz, aptt, tz, protein c, tat-complexes, fi+2 and d-dimer were measured. results d-dimer levels increased steadily between day 0 and day 7. tatcomplexes showed a slight increase from day 0 (2.4 bg/i) to day 3 (15.3 ~tg/i). on day 7 tat levels were down again (2.0 p,g/l). fl+2 (day 0:1.0 ng/ml) also showed a slight increase on day 3 (1.3 ng/ml). protein c decreased steadily from day 0 (108%) to day 7 (15%). conclusion during the initial phase of oac therapy a coagulation activation is reported but no significant elevation of tat or fl+2 was found. this result shows that additional heparin and asa therapy was sufficient to avoid systemic coagulation activation. the increase of d-direct should be interpreted as a si~=m of local fibrinolytic reaction due to stent implantation. three methods for the determination of prothrombin time from capillary blood in patients under oral anticoagulation have been investigated. two methods were run on coaguchek® monitors (boehringer mannheim) from capillary whole blood. after fingerpuncture the first drop of blood was applied to the well of a coaguchek® test strip directly from the finger-tip, whereas the second drop was sucked into a non-anticoagulated plastic capillary (hirschmann) and immediately applied to the test strip -and vice versa to eliminate any influence of first and second drop of blood. the third method was hepato quick (boehringer mannheim) which was determined out of citrated capillary blood from an earlappuncture. 66 specimen of patients under oral anticoagulation were investigated. the method comparisons between each of the coaguchek® methods and the laboratory method show good results and the correlation between the coaguchek® methods is excellent. mean differences to the lab methods are -0.1 inr in both cases. no mean deviation was detectable between the coaguchek® methods. scattering of coaguchek® versus hepato quick was +/-0.6 inr in the range 1 to 4 inr except for three outliers and one patient with fluctuating results in the lab method which could not be resolved. introduction: haemorrhagic coumarin skin necrosis is a severe complication during initial phase of oral anticoagulant therapy. histological examination shows thrombotic occlusion of small vessels, but little is known concerning the pathophysiologic background of the bleeding component. recently, we described protein z deficiency in patients with bleeding complications of otherwise unknown origin. thus, we were prompted to measure protein z in patients with coumarin skin necrosis. patients: 4 patients (i man, 4 women; age: 35±10 years) suffering from haemorrhagic coumarin skin necrosis were examined. all patients had normal liver protein synthesis function, none was under oral anticoagulant treatment during this study. method: protein z antigen test, diagnostika stago, france. results: 4 out of the 5 patients examined had diminished protein z levels ( 700, 820, 1080, 1700 ug/l) in comparison to normals (2900 ug/l). in one of our patients, protein z was normal (3020 ug/l). conclusion: low protein z levels are additional risk factors for haemorrhagic coumarin skin necrosis. oral anticoagulant therapy is the treatment of choice in patients with need for long-term anticoagulation. since oral anticoagulants interfere with the function of vitamin k, it is not clear whether stable oral anticoagulation can be achieved in patients with need for continous substitution of fat-soluble vitamins including vitamin k. we report about a 59-year-old man who had experienced progressive hypertrophic obstructive cardiomyopathy over the preceeding 21 years. atrial fibrillation has been first diagnosed 18 years ago. latter on, recurrent ischemic attacks and embolism of the right arteria iliaca occurred. in 1993 the patient received extirpation of the ileum and subtotal amputation of the jejunum because of mesenteric infarction. the resulting short bowel syndrome requires continous substitution of fat-soluble vitamins. since vitamin k free preparations of fat-soluble vitamins for parenteral use are not available, prophylaxis of thrombosis has been performed with unfractionated hepadn. as a consequence of the longterm treatment with hepadn the patient developed severe osteoporosis. therefore, the decission 1:o discontinuate heparin therapy and initiate oral anticoagulation has been made. because of its shorter halflife warfarin (coumadin) was used instead of dicoumarol. over a 4 weeks lasting induction phase inr values were controlled daily. a dosage regime starting with '10 mg warfarin at the day of vitamin application (day 1) followed by 3.75 mg on day 2 and 1.25 mg on days 3, 5, and 6, respectively, was found to be optimal to maintain inr values within the target range (inr: 2.0-3.0). in order to minimize the risk of hemorrhage the vitamin administration was changed to the subcutaneous route. during an observation period of 6 months neither any bleeding or thrombotic complications nor a vitamin deficiency occurred. these data indicate that stable oral anticoagulation can be achieved despite extreme variation of vitamin k plasma levels. portable monitors for home monitoring of inr are well established for adults on oral anticoagulants. patient's compliance is improved as well as long term outcome. experience concerning accuracy of the procedure in children is limited. 32 inr determinations were performed in parallel from venous and capillaryblood samples of an infant on phenprocoumon, starting at the age of 4 months. the coaguchek® monitor from boehringer mannheim was used. choosing an arbitrary range of agreement of ,qnr 0.5 for both determinations, 81% of the measurements were within the defined range. 5/6 outliers were due to low inr resulting from difficulties in capillary blood sampling. the degree of agreement increased when the procedure was performed at least once a week. in conclusion: inr determination with a portable monitor may be helpful in home monitoring oral an.ticoagulant therapy in young children. a dose adjustment should be done only on the base of inr determination of venous blood -if it is considered the gold standard -to avoid over-anticoagulation. a stable anticoagulation is one of the most difficult tasks in attending patients with heart-valve-prosthesis. if prothrombin times are out of the therapeutic range, the risk of bleeding or thromboembolism increases disproportionately. for this reason any improvement in anticoagulant control and/or management can have far reaching consequences in decreasing complications, in extending longevity and in improving quality of life. for the first time a clinical trial was started in 1986 and continues until today at the cardiac rehabilitation center bad berleburg, germany with patients mainly after heart valve replacement. the patients were trained to measure their own prothrombin time and to adjust their own dosage of the oral anticoagulant. within six years 600 patients were trained: 216 patients could be followed up with regard to their selfdetermined prothrombin times. the results were within the therapeutic range in 83.1% of the measurements (n=14.812) taken by the patients themselves. on average, the patients who determine their prothrombin time themselves did so at a weekly interval. neither major bleeding nor thromboembolic complications could be observed in the 205 patient-years of home prothrombin estimation. it is to be hoped that the usual rate of complications can be reduced when patients determine their prothrombin time themselves at a close interval, resulting in more constant values in the therapeutic range and slight corrections of the anticoagulant dose. home prothrombin estimation promises better quality of life and has a considerable potential to achieve this goal. circulating plasma thrombomodulin (tm) is a novel endothelial cell marker, which may reflect endothelial injury. tm acts as thrombin receptor which neutralises the fibrin-forming effect of thrombin, and also accelerates the formation of the anticoagulant protein c/s pathway. tm therefore belongs to the anticoagulant defence system against thrombosis. increased tm levels have been described in various diseases such as ards, thromboemboembolic diseases, ttp, diabetes, le and cml reflecting alterations of the vascular system at the endothelial level. to find out to what extent cardiac catheterisation imtates vascular endothelium, tm concentrations (stago, asnieres, france: x 10 3 iu/ml) were investigated prospectively in 58 infants and children (three days -16 years). blood samples were drawn before the intervention, immediately at the end and 24 h later, snap frozen (-70 °c) and investigated serially in dublicate six weeks -3 months later. the results (median and range values) are shown in the enhanced tm concentrations immedately after the operative intervention, followed by normalisation within 24 h, indicates that cardiac catheterisation in pediatric patients rather leads to a short lasting irritation of the vascalar endothelium than to severe irreversible endothelial damage. recently in an al=wl" based method dahlb~ick et al described in vitro resistance to the anticoagulant effect of activated protein c (apc) in thrombophilic adult patients. apcr is in the majority of cases associated with the arg 506 gin point mutation in the factor v gene. concerning the special properties of the neonatal hemostatic system (low vitamin k dependent coagulation factors, physiological prolongation of the pt and aptf) we adjusted this ap'it based method (chromogenix, m~,lndal, sweden) to neonatal requirements: apcr was measured in 120 healthy infants according to dahlb~ck. the results were expressed as apc-ratios: clotting time obtained in a 1:1, 1:5 and 1:11 dilution with factor v deficient plasma (instrumentation laboratory munich. germany) using the apc/caci2solution divided by clotting time obtained with cac12 in the same i:1, 1:5 and 1:11 dilution. in addition, plasma of 24 neonates with septicaemia were investigated and data of 18 infants aged birth -three months with arg 506 gin +/-were shown. the arg 506 gin mutation of the factor v gene was assayed by amplification of the dna samples by pcr followed by digestion of the amplified products with the restriction enzyme mnl i. results were confirmed by sscp -analysis or by direct sequencing of dna from patients with apcr. results are shown in the 1.6(1.4-1.95) neonates and infants were considered to be apcr when the aptt ratio was < or = 2. concerning the special properties of the neonatal hemostatic system, our data show concordance with the pcr method in neonates and infants only, when the aptt based method was performed in the i: 11 plasma dilution. case report: we report on an 8-year old boy with severe hemophilia b and frequent screaming at night. eeg showed spike wave activity, starting from the temporal lobe, but generalizing within seconds. complex partial seizures were diagnosed and therapy with carbamazepine was initiated. as no improvement was seen nmr was performed. this revealed lesions within the right frontal cortex. higher doses of carbamazepine were not succcssfull as was therapy with phenytoin and pfimidone respectevely. the patient is now treated with carbamazepine and valproate. he still suffers from one short seizure per day. because of his seizures we started prophylactic replacement therapy with 600 i.e. factor ix twice per week. discussion: in 1992 wilson et al. first detected brain abnormalities in 25 of 124 children and adolescents with hemophilia a or b who were negative for immunodeficieney virus (1). the most common findings (14/25 patients) were small, focal, nonhemorrhagic white matter lesions of high signal intensity on t2weighed images. similar lesions have been reported in children with sickle cell cerebral infarction (2) . only three of these 14 patients had seizures, all of those having a documented history of intracranial hemorrhage. our patient has similar lesions as those described by wilson et al. but no history of intracranial hemorrhage is documented. even if tuberous sclerosis might be a differential diagnosis, we think that the abnormalities are related to hemophili a or its treatment, because the patient has no further signs of this disorder. conclusions: 1. in patients with hemophilia and seizures nmr might be useful as a high sensitive method for the detection of gray and white matter changes. 2. further studies should be initiated to determine the prevalence of pathological conditions in the brain of hemophiliac patients. disseminated intravascular coagulation (dic) is a rare, but foudroyant disease occuring in gram-negative sepsis like meningococcal septicemia. despite the avallibility of potent antibiotics, mortality in mertingococcal disease remains high ( about 10 % ), rising to 40 % in patients presenting with severe shock and consecutive dic. as the clinical course and the severity of manifestations of systemic meningococcal infections varies there is a need for early diagnosis of the infection and stage of coagulopathy in order to reduce the high mortality rate. few and rapidly available parameters are needed to classify the wide spectrum of clinical and laboratory findings in patients with dic. the parameters include partial thmmboplastin time, pmthmmbin time, plasma levels of fibrinogen, fibrin monomers and dimers, fibrin degradation products and the thrombocyte count. monitoring the course of hemostaseologicai findings in 26 pediatric patients with systemic meningococeal infections we observed a change of coagulation parameters as early as in the first stages of the infection: a prolongation of partial thromboplastin time to an average of 69. 1 sec (range 22 -150 sec, norreal 30 -45 sec), a decrease of prothrombin time to 45.7 % (range 13 -71 %, normal 70 -100 %) and of antithrombin iii to an average level of 16. 8 u/ml (normal 20 -29 u/ml ) was found 1 to 4 (-6) hours after admission. the consecutive development of hemostaseological parameters mentioned above permitted to define the stage of coagulopathy and thus to induce a stage related therapy. primary treatment consisted in control of shock by liquid substitution, compensation of metabolic acidosis, correction of clotting disorders ( at iii and heparin in stage of pre-dic ; at iii and fresh frozen plasma in case of advanced dic ) and treatment with g-lactam antibiotics ( e. g. cefotaxime or ceftriaxone ). an early assessment of the coagulation disorders in meningococcal disease can be based on few coagulation parameters, thus an appropriate treatment may be arranged to prevenl the patient from a fatal outcome of meningococcai septicemia and protect him from the development of a waterhouse-friderichsen-syndrome. this study was designed to prospectivdy evaluate coagulation and flbrinolyfie activation in 60 children (neonate -16 years) during cardiac catheterisation with low dose flush heparin (10 iu/ml saline). aptt (instrumentation laboratory: see), anti xa activity (xa; chromogenix: iu/ml), prothrombin fragment ft.2 (f1.2; behring werkc marburg: nmol/l) and d -dimer formation (d-d; bnhring werke/vhrburg: ug/l) were investigated before (t1), at the end (t2) and 24 h after cardiac catheterisation (t3). in addition, to evaluate the influence of inherited thrombophilia in all patients resistance to activated protein c (apcr), protein c, protein s and antithrombin were investigated. during catheterisation median (range) hepadn was administered in a total dose of 60 (17-206) iu/kg bw. in addition infants < 6 months of age (arterial catheterisatiun only) or patients with known thrombophilia received 300 -400 iu/kg hepafin for fmther 24 hours. the results (median and range) are shown in the ft.2 was sigificanfly elevated above the pediatric boundary immediately after the intervcation and nearly reached baseline values 24 h later. in contrast no cfinically relevant fibrinolytic activation was seen: d -dimer formation increased within the pediatric boundary immediately after the catheter and returned to basdine levels 24 h later. three children showed resitance to apc. tn one child stroke occurred before. not knowing the result of apcr in the remaining two patients only one neonate received further prophylactic heparin. the third neonate without heparin prophylaxis suffered from venous occlusion within two days after the intervenfon~ in addition, no protein c, protein s or antithrombin deficiencies were found. although administration of low dose flush heparinisation during cardiac cathetefisation could not prevent short -term coagulation activation, no thrombotic events occurred in children without inherited thrombophilia. if fnrther prophylactic hepariuisation in children with a~r, protein c, protein s or antithrombin deficiencies may prevent vascular occlusion requires a more intensive study. a.sandvoss, w.eberl, m.b0rchert introduction: capillary leakage, edema and hypovolemia are common complications in preterm infants especially if birth weigth is below 1.500 g. septicemia, asphyxia and immaturity seem to be most important risk factors. to determine the influence of c 1-esterase inhibitor (cilna) in preventing contact phase and complement activation we investigated c11na concentrations in normal and symptomatic preterm infants. methods: activity of cilna were measured by chromogenic substrate method (behringwerke), cilna concentration with radial immunodiffusion (behringwerke,germany). results: cllna-activity in asymptomatic preterm infants (n= 14) was 65+/-15% of normal at birth. healthy newborns showed activities of 80+/-20%. cilna reached normal adult values 2-4 days after birth. preterm infants with respiratory distress syndrome(n = 14) showed lower activity on day 2-5, patients with additional septicemia (n=15) had decreasing c1 ina-activities in the first three days of life. individual course of cllna-activity and thrombocyte count correlated in the group with irds with and without septicemia. in children with capillary leakage onset of diuresis went parallel with raising cllna-activity. markers of contact phase (f xlla) and complement activation (c 5al were investigated in single cases and evidence for involvement of both systems was found. conclusion: contact activation and complement system play an important role in capillary leakage in preterm infants. cilna regulates both systems. activity of cilna correlates with clinical course, substitution therapy is possible and may improve outcome of these critical ill patients. antiphospholipid antibodies (apa) interfere with hemostasis probably by inhibition of protein c or prothrombinase complex. thereby, apa might lead to thrombosis or increased bleeding. however, incidence and clinical importance of apa has not yet been investigated in children. therefore, we assayed plasma samples of 220 children, aged 0,1 to 19 years (mean 7 years) by elisa detecting igg-and lgm-antibodies directed against eardiolipin, phosphatidyl serine and phosphatidic acid. in patients with increased bleeding, thrombophilia or prolonged clotting tests a detailed coagulation analysis was performed. according to their diagnosis children were devided into 5 groups: i. autoimmune diseases, ii. infections, iii. metabolic diseases, iv. other diseases, v. healthy children. results: apa were found in 69/220 patients. in the respective groups we demonstrated apa in the following proportions: 1. lgg-isotype: activitiy of c1 esterase inhibitor (c11na) is reduced in preterm infants especially if birth weigth is below 1.500 g and respiratory distress syndrome and/or septicemia is present. capillary leakage with generalized edema, hypovolemia and hypotension is resulting in imbalance between inhibition and activation of contact phase and complement system. iln four patients we investigated seven courses of substitution ;with commercial c1 esterase inhibitor preparation (berinertr,behringwerke), case reports are given. all patients had clinical symptoms of capillary leakage, all had septicemia accompanied by either respiratory distress, disiseminated intravascular coagulation or mutiple organ failure. jefficiacy of substitution therapy is dose related, supranormal iactivities of cilna are necessary, reflecting raised consumption of inhibitor in ongoing disease. clinical effects on diuresis, catecholamine need and especially on thrombocyte counts are demonstrated. or arterial thromboembolic event in children e. lenz, c. heller, w. schr6ter*, w. kreuz johann w. goethe-universit~itskinderklinlk, frankfurt a. main, germany * georg-augast-universit/itskinderidinik, g/3ttingen, germany venous thrombosis as well as arterial thrombo-occlusive events are rarely observed in childhood, but can lead to life-threatening situations and longterm sequelae in these patients. after the initial stage of treatment (thrembolysis or thrombectomy) the pediatrician has to decide how to efficiently prevent re-thrombosis in the individual patient. anticoagulation after venous thrombosis is generauy recommended for 6 months after the event; if an underlying thrombophilic condition has been detected in the patient anticoagulation has to be considered lifelong. when evaluating antithrombotic therapies for children it is of importance to consider whether the anticoagulatory effect is mainly necessary in the venous or arterial vessel system. the hemorrhagic risk and side effects of the different anticoagulatory preparations have to be taken into account, especially when treating small children. only limited experiences exist concerning the suitability of the preparations for long-term anticoagulation in children and general recommendations on the ideal dosage in pediatric patients are still missing. we want to disscuss different types of anticoagulants (such as coumarins, unfractionated heparin, low molecular weight heparin (lmwh) and inhibitors of platelet aggregation) their mode of action, their suitability for pediatric patients and their side effects and relevance of these side effects especially in children. from the experience in our own pediatric patients, we would like to report on the indications, which can be given to administer these different preparations, the dosage regimen we recommend and the laboratory tests to monitor save and efficient re-occlusion prophylaxis in our patients. in this context we would like to present our data on 8 patients with either thrombosis or arterial infarction due to a thrombophilic condition, who had all contraindicatioas to oral anticoagulation by coumarins. because prophylaxis for re-thrombosis was mandatory in these patients, lmwh was given for long-term anticoagulation in a dally subcutaneous dosage of 100-150 anti-xa u/kgbw. monitoring was done by anti-xa-test (0,4-0,8 anti-xa u/ml). under this regimen none of the patients developed re-thrombosis or bleeding complications. alopecia was seen as a side-effect. this study was designed to prospectively evaluate coagulation and fihrinolytic activation after cardiopulmonary bypass with aprotinin (2x17000 u/kg bw) in 42 infants and children aged 0.1 -15 years, and to correlate these findings to the clinical outcome. prothrombin fragment f 1.2 (f1.2; behring werke marburg: nmol/l), antithrombin-serinesterase -complex (atm; stago: ng/ml), d -dimer formation (d-d; behring werke marburg: ug/l), tissue-type-plasminogen activator ag (t-pa; chromogenix: ng/ml), plasminogen activator inhibitor 1 antigen (pai; chromogenix: ng/ml) and cl-inhibitor (c1; behring werke marburg: x 10-3 g/l) were investigated before the operation (t1), at the end of the operation (t2), and on postoperative days 1 (t3), 4-6 (t4) and 7-9 (t5), respectively. the results are shown in the table (median and median absolut deviation): t1 t2 t3 t4 "1"5 nv fi.2 0.9 +/-0.5 1.7 +/-0.9 1.4+/-1 1.8+/-0.8 1.6+/-0. the platelet (pl) function defect induced by thrombolytic agents has been attributed either to the degradation of pl surface receptors or to the anti-aggregatory effect of fgdps. in contrast to other plasminogen activators scu-pa is intimately inked with pl: they can rapidly incorporate exogenous seu-pa, release it upon stimulation and bind the proenzyme. recently we have reported that exposure of prp to recombinant scu-pa (2.5-t00 um) in timed interval 1-30 min resulted in dose-dependent inhibition of pl aggregation. timecourse changes of the process were followed by the biexpotential kinetics: a rapid initial inhibition during the first 3-5 rain with the moderate suppression of pl aggregation in the 30 min period. when tcu-pa (25-100 nm) was exposed to prp in the same conditions dose-and time-dependent inhibition of pl aggregation was also observed. since the effect was obtained no earlier than t0 min after exposure of tcu-pa to prp, and the threshold dose was higher. comparable inhibition of pl aggregation was obtained with 25nm of scu-pa versus 100nm of tcu-pa and the llbrinogen depletion by the end of the 30 min period was 2% and 30% respectively. it's likely that tcu-pa and its precursor have different mechanisms of action on the pl aggregatory function. in a recent study we have shown that recombinant rscu-pa inhibits platelet (pl) aggregation in prp. to exclude the possible influence of rscu-pa/plasma interfere on this process the aggregation of washed pls was under the investigation. pls were washed according to modified mustard's method, suspended in buffer and adjusted to 250,109/1. the resuspended pls were exposed to 5-100 nm of rscu-pa for 30 min at 370(;. at time points 3, 5, 15 and 30 min the aggregation with 0.6 iu/ml of thrombin was measured. it was found that the exposure of pls to rscu-pa (20-100 nm) for 3 man resulted in marked inhibition of their aggregation. since after 15-30 man of incubation with 20-50 nm of rscu-pa the inhibitory effect on pl aggregation became less pronounce or even disappeared. when 5 nm of rseu-pa was used the inhibition of pl aggregation became significant only by 15 rain of exposure period and didn't change for 30 man of investigation. the observed results may be cormeeted with uptake of rscu-pa by pls from surrounding buffer as well as with individual variations of pl response to the same concentration of rscu-pa. loss of glycosylation may result in a reduced platelet (p) survival and perhaps altered function. we analyzed the structural and functional effect of specific deglycosylation (combinations of n/o-glycosidase and neuraminidase treatment) of p and isolated p gpib. washed and formaldehyde-fixed p were digested as follows: 1) with neuraminidase (0.125u/ml) + o-glycosidase (3.1mu/ml) + n-glycosidase (1.25u/ml), 2) with neuraminidase alone (0.2u/ml), 3) with n-glycosidase (2u/rnl) and 4) with neuraminldase (0.2u/ml) + o-glycosidase (5mu/ml). all reactions were performed in the presence of protease inhibitors (pmsf, leupeptin, sbti), after washing x2 the p and identically treated controls were analyzed by flowcytometry with the antibodies 6di (mab: a-gpib), 7i-l2 0vlab: a-gpiiia), and the lectins wheat germ agglutinatinln (wga, for neunac) and peanut agglutinin (pna, for [3dgal(1-3)-galnac) which confirmed effective and specific deglycosylation by the respective enzymes (but gave only minor differences with 6di and 7h2). the botrocetin (13) and ristocetin (r)induced agglutinations showed arer treatment 1) (all enzymes) a full inhibition of r-induced agglutination but only a mildly reduced b-induced agglutination (70% of normal). treatment 2 and 3 (neuraminidase alone, and n-glycosidase alone) affected both agglutinations only mildly (70-80% of normal).treatrnent 4) (o-deglycosylation) however showed a major inhibition of r-agglutination down to 30%, while b-agglutination interestingly was almost fully retained. the results of the rotary shadowing electron microscopy of purified gpib suggested a collapse of the normally stretched, glycosylated, gplb, not only after the treatment with all three glycosidases, but also .after o-deglycosylation alone. we conclude that oglycosylation is most important for ristocetin-induced platelet-von willebrand factor-interaction and responsible for the typical stretched shape. the phenomenon of in vitro platelet aggregation and consequent pseudothrombocytopenia (ptcp) in the presence of calciumchelatization by na-edta and sodium-citrate was studied in blood samples of a patient. initial platelet counts electronically measured were 20000/ul blood anticoagulated with na-edta and sodium-citrate. normal platelet counts were found in heparin-anticoagulated blood and in capillary blood. immunoglobulines of the igg and igm subclass were identified in the patients plasma. by incubation of the patient's serum with platelets of healthy individuals, platelet-clumping occurred in the presence of na-edta and sodium-citrate but not in the presence of heparin. the platelet membrane glycoproteins (gp) hb/llia, ix and iiia/vnr g-chain were involved in the antigen antibody reaction as demonstrated by specific antibodies and flow-cytometry. on platelet surface permanent calcium-exchange and -replacement is dependent on external calcium concentration. calcium depletion induced by calcium chelators as na-edta and sodium-citrate might conformationally change platelet surfaces and induce formation of neoantigens. the decrease of gp llb/illa platelet surface antigen to 10% (normal >75%) indicated the important role of the gp iib/iiia receptor at ptcp. the saliva of tdatoma pallidipennis, a triatomine bug, was found to contain a protein called "pallidipin", that specifically inhibits collageninduced platelet aggregation but not adhesion or shape change. to investigate the mechanism of action of recombinant pallidipin the influence on platelet fibdnogen binding after activation by collagen type i in different concentrations was measured by flow cytometry. the same concentrations of pallidipin that inhibited the couagen-induced platelet aggregation completely did not cause any inhibitory effect on fibdnogen-binding in the prp from the same donor measured contemporaryly. collagen type i-induced platelet aggregation of cd36-deficient platelets from two different unrelated blood donors was inhibited by the same concentration of pallidipin that inhibited aggregation of control platelets. there was no inhibition of collagen-induced fibdnogen-binding in the cd36-deficient platelets as well. pallidipin did not cause inhibition of collagen-induced membrane expression of cd62 and cd63 of control and cd36-deflcient platetets as measured by flow cytometry. however eadier studies had shown an inhibition of collagen-induced atp and {3tg secretion by pallidipin. therefore we compared the effect of pallidipin in unstirred and stirred prp samples. while pallidipin had no effect in unstirred samples it showed strong inhibition of ptg secretion in stirred samples. we therefore conclude that pallidipin does not act on collagen-induced aggregation through cd36 and that the inhibition is a post fibdnogenbinding event. pallidipin does not influence the first steps in secretion, which are independent from cytoskeleton and platelet-platelet contact, but inhibits the following steps. 17-hydroxy-wortmannin does not inhibit the transport of 1nm-gold labelled fibrinogen in resting platelets. e. morgenstem, b. kehrel and k.j. clemetson medical biology, saarland univ., homburg, germany, haemostasis research, univ. muenster, germany and theedor-kocher-lnstitut, univ. bern, switzerland. wortmannin, an inhibitor of phosphoinositide 3-kinase and of myosin light chain kinase blocks reactions of the activated platelet. to obtain informations about the role of the contractile cytoskeleton in receptor-mediated transport of resting platelets, the effect of 17-hydroxy-wodmannin (hw) on the endocytosis of fibrinogen from the surface of resting platelets was studied. gel filtered platelets (gfp) were incubated for 10 min at 37°c with hw (3x10-6m) or with iloprost. controls and gfp preincubated with hw or ilopmst were incubated with 1.4nm-gold labelled fibrinogen molecules (fg-au; final concentration 40p.g/ml) at 37°c. the experiments were stopped after 5 or 30 min by rapid freezing. after freeze substitution in acetone with 4% osmiumtetroxide, sedal sections were prepared. the sections were examined after incubation with ascorbic acid (5% in h20) for 30 rain at 20°c (to reduce metallic osmium) and silver-enhancement using danscher's (1981) method (to visualize the fg-au). examination of adp stimulated platelets in the presence of 40fg/ml fg-au shows that the ligand is able to mediate aggregation. the examination reveals, that fg-au was present in a low density on the platelet surface, in higher density in the surface connected system (scs), in coated pits and vesicles and separated smooth vesicles (representing endosomes?) as well as in the matrix of alpha-granules. after 30rain, the number of labeled granules was increasing. labels on the surface and on the mentioned cytoplasmic membranes were observed during the whole period of incubation. hw or iloprost did not alter the resting gfp and the mentioned qualitative ultrastructural findings in both preparations did not show differences to the controls. we conclude from the results with hwthat the regular contractile function of the cytoskeleton is not necessary to transport the fg-au in resting platelets. methods: edta anticoagulated whole blood was incubated with thiazole orange and analyzed with a flow cytometer. young platelets were defined by having a high fluorescence from thiazole orange (normalized to platelet size). platelets were also incubated with fluorescent antibodies to gpib, gp lib/ilia and gmp-140 (two colour method). results: surface expression of gpib was the same in young and older platelets. results for gp lib/ilia and gmp-140 (in resting and activated platelets) will be presented. conclusion: young platelets can easily be detected using thiazole orange and flow cytometry. there is no differential expression for gpib. further results will be presented. the influence of erythrocyte and thrombocyte content on the release of atp by different agents in whole blood specimens was tested. the measurement had been performed in the lumi-aggregometer using the principle of the luciferin-luciferase reaction. altogether 39 blood samples were diluted gradually before induction of the release reaction by arachidonic acid (1,25 mmol/i final concentration), adp (30 ijmol/i) and collagen (1,0 and 5,0 tjg/ml). the peak of the obtained curves was transformed into percent values of the maximal deflection by the undiluted sample (= peak in relation) and into atp concentrations (= absolute peak) after testing the atp standard in parallel for each dilution step separately. the peak in relation increases by increasing dilution with all inducers. it was identic with the atp standard and with collagen, somewhat lower with arachidonic acid and much higher by adp. a luminescence-optical effect may influence all these results. the absolute peak decreases by dilution under arachidonic acid and collagen as it was expected by the decreasing thrombocyte content of the samples. under induction by adp no decrease of the absolute peaks by increasing dilution of the samples was abserved. this can be explained only by liberation of atp from the erythrocytes. the atp standard is essential for the quantification of the release reaction. adp doesn't suit for it. collagen with a final concentration of 1 pg/ml was proven as the best inducer. platelet aggregation induced by several agents has been photometrically investigated in disc shaped rotating cuvettes coated with vessel wall tissues obtained from human umbilical cord, either endothelium or smooth muscle cells or extracellular matrix or combinations of them. in addition, effects of endothelium incubated with several cytokines on platelet aggregation have been studied. endothelial cells strongly inhibited aggregation depending on their cell count and the concentration of the inducer. smooth muscle cells showed the same effect but very less marked. in presence of extracellnlar matrix spontaneous aggregation occured. endothelium could inhibit this spontaneous aggregation when present in the same cuvette, smooth muscle cell could not. incubation of endothelium with several cytokines increased its anti-thombotic properties. for example, at a platelet count of 3x105/id in the prp, 10 -6 m adp led to maximal aggregation in uncoated cuvettes, in presence of 5,5x106 endothelial cells aggregation was completely abolished, in presence of 2,75x10 "6 cells aggregation was decreased to 40%. smooth muscle cells diminished the aggregation effect of 0,1 nih thrombin to 67% when only one side of the cuvette was coated and to 63% when both sides were coated. endothelium could not inhibit aggregation induced by 2,5 x 10 -6 m adp but endothelium incubated with 500 u/ml tnf-a or 30 u/ml intedeukin-lfl or lmm l-nitro-arginin for 24 h did completely inhibit aggregation. platelets become sticky and adhere to surfaces or to another without contracting and secreting. during maturation of megakaryocytes finally platelets lost their genomic nuclear message. only mitochondrial dna of platelets can be identified. we focused our attention on the impact of mitochondrial dna and the mitochondrial transscriptive mechausisms during platelet activation in normals. materials and methods: leucocyte free (nagentte chamber, flow cytometric analysis) platelet rich plasma or platelet concentrates a_~er hemapheresis were filtered by pall 100 leucocyte filters. the influence of different anticoagulants (commercially available sarstedt tubes containing citrate, heparim edta and 500 atu/ml hirudin wacker) was examined. activation was due to a 60 nun. hemapheresis procedure ( 3-5fold increase of cd 62, cd 63) and ex rive stmaulation due to 4 niy u/ml thrombin, 0.025 m cac12 or combmatious. the guanidiurn method for total rna preparation were used according to t. brown: current protocols in molecular biology 4.21-4.9.14,1991. different primers of mitochondrial genome (e.g. cytochrome b and atpase) were prepared using pcr and mitochondrial transscription was examined using northern-blot-technique. results: 1., there is less activation of mitochondrias using hirudin anticoagnlation, but a 2fold increase of mitochoindrial rna content in heparinized samples. 2., stimulation with thrombin leas to an increase to 5.5 e-l0 rna btg/platelet, compared to 4.7 -4.8 e-10 rna ~tg/platelet under unstimulated conditions.. conclusion: there is evidence for the importance of platelets mitochondrial dna and mitochondrinl transsefiption in regulation of cytosceleton and platelet activation. thrombospondin-1 (tsp-1) is a large homotrimeric glycoprotein originally identified as a platelet alpha-granule component. the investigation of its putative role in a variety of pathophysiologies like haemostatic disturbance, malignancy and wound healing requires specific laboratory reagents. monoclonal antibodies are one of the most powerful of these reagents. therefore, we purified human tsp-1 from thrombin-stimulated platelets using affinity chromatography to generate monoclonal antibodies in mice. a subclass igg 1 monoclonal antibody designated 48.42 was purified from ascitic fluid and further characterised. western blot experiments demonstrated that this antibody reacted only with the unreduced molecule whereas the tsp-1 subunit chain was not recognised. no cross-reactivities with human fibrinogen, fibronectin, vitronectin and von willebrand factor were found. preliminary results indicate that the monoclonal antibody 48.42 can be used to investigate tsp-1 function in several assays including immunocytochemistry and cell adhesion as has been demonstrated for hl-60 cells. in addition, a sandwich enzyme immunoassay was developed using goat-antihuman tsp-1 igg and derivatised monoclonal antibody 48.42 (peroxidase, biotin) as a sensitive method for detection of tsp-1 in human body fluids. in the following study the expression of the platelet antigen (cd62p) and the leukocyte antigen (cdllb) were measured in whole blood, in addition to platelet-leukoeyte adhesion (rosette formation) by means of multicolour fluorescent labelling (cd45, cd14, cd42a). the measurements were carded out both in freshly drawn whole blood which had been antieoagulated with different agents, and in stirred samples of whole blood under controlled conditions (37°c, 1000 rpm, different stirring times). the results are presented as the percent positive events in each gate (platelets, leukocytes -pmnl, monocytes, lymphocytes and rosettes -plateletpositive events in the pmnl, monocyte and lymphocyte gates), whose mean fluorescence is given in addition to an index comprising the product of the percent positive events and their mean fluorescence. stirring (max 15 rain) induced an increase of cd62p on the platelet surface of ca. 10%, without any change in the mean fluorescence. under these conditions increased cdllb on pmnl and monoeytes could be detected. an increase in the rosette formation could also be measured (greater index), in that the percent of monocytes which were platelet-positive increased with no change in the mean fluorescence of the positive events, whereas pmnl showed an increased mean fluorescence, but not an increased number, of platelet-positive events. the time-dependent changes in rosette formation on stirring could be further increased by addition of adp. these results show that it is possible to measure rosette formation, and also the influence of effector agents (inhibitors or activators of platelets or leukocytes) on rosette formation, in whole blood using flow eytometry. 17 itp patients undergoing splenectomy were observed after 1-30 years following operation and divided into 2 groups. first group consisted of 8 patients with normal platelets count and absence of haemorrhagic syndrome. second group was formed of 9 itp-patienfs with episodes of thrombocytopenia recovery following certain time period after splenectomy. in the aim to study the cellular immunity there were carried out immunophenotypical investigations of blood samples using immunofluorescence method with monoclonal antibodies application. the increase of b-cells, expressing cd22, cd37, hla-dr-antigen has been revealed in the 2nd group. quantity of srfc, cd3 +, cd5 + cells in the blood of recovered patients was lower than in patients of the first group. this group was also characterized by statistically significantly increased level of cd4 + cells while the cd4/cd8 ratio was equal to 1.0 :i: 0.3 % (0,5 + 0,1% in patients of the second group, respectively, p>o,05}. also the relatively high expression of activating antigens in patients with thrombocytopenia recovery after splenectomy was stated. among infectious complications in all patients observed were predominantly found various types of throat infection, mainly with unsatisfactory treatment possibilities. we have observed the opsi-syndrome in 2 patients, being featured with marked tiredness, breath loss, intolerance of hard physical working, diminished ability to maintain physical activity. extracellular matrix (ecm) produced by human endothelial cells closely resembles the vascular subendothellal basal lamina in its organization and chemical composition. thus it contains collagens, fibroneetin, von witlebrand factor, thrombospondin, fibrinogen, vitronectin, laminin and heparin-sulphate. platelets carry different receptors on their membrane surface with specific binding capacities for one or more of these extracellular matrix proteins, such as glycoprotein (gp) iibiiia, gp ib/ix and gpiiib. incubation of platelets with ecm results in platelet adhesion, degranulation, prostaglandin synthesis and aggregation. we studied patients whose platelets showed either a receptor defect in gpiibiiia or gpiiib or a storage pool disease. adhesion experiments were performed using siliconised glass, collagen coated surfaces, immobilized fibrinogen as well as human subendothelial matrix. platelet adhesion of patients with thrombasthenia glanzmann (receptor defect of gpiibiiia) resulted in a total lack of binding to silieonised glass and immobilized fibfinogen. adhesion to collagen was almost normal in spite of the fact that only single platelets sticked to the surface and no microaggregates were observed. the adhesion to ecm was diminished and also no aggregates were detected. patients with a receptor defect in gpiiib showed normal platelet adhesion to siliconised glass and immobilized fibrinogen but binding to collagen and ecm was markedly reduced, while platelets with a storage pool defect sticked to siliconised glass but failed to adhere to ecm. by centrifugation of citrate blood (250 x g, 10 min) erythrocytes and leucocytes go to the bottom, whereas plasma and thrombocytes stream in the upper part of the probe. so the thrombocyte count doubbles in the platelet rich plasma in contrast to the platelet count in the whole blood volume. if the thrombocytes are more or less activated, they adhaere on erythrocytes, leucocytes or aggregate end are not able to stream upwards. the quotient between thrombocyte counts in prp and whole blood is a measure for thrombocyte activation. we chequed the value of this screening in different groups of patients with arterial occlusions disease (aod), chronical venous disease (cvd), diabetes mellitus (dm] and in healthy control persons (control). variation coefficient of the method is 3.7 (prp) and 4.4 (tc) respectively (coulter counter). differences to the control group are significant. changes in the patient groups in dispensaires follow up 5 years are also significant. nicardipin -induced immunthrombocytopenia p. eichler 1, c. hinrichs 2 , g greinacher l i.institut fur immunologic und transfusionsmedizin, ernst-moritz-arndt-universitat greifswald, 2. deister-s0ntel-klinik, bad m0nder drug-dependent immune-thrombocytopenias are a rare but clinically important variant of immune-thrombocytopenias. patients are at risk to suffer from severe bleeding complications. especially in patients receiving multiple drugs, diagnosis of drug-dependent immune-thromboeytopenia is often difficult. we report the case of a 71 year old male patient who received allopurinol, captopril, digitoxin, furosemid, and nieardipin. the patient presented with hematomas (pit. count < 10 g/l) and later developed bone marrow dysplasia. in an elisa using whole platelets and patient serum, a weak reactivity in the presence of furosemid, but a stronger reactivity in the presence of nicardipin (antagonil, ciba-geigy) could be demonstrated. the reaction pattern is given in the the enzyme-immunological determination of soluble fibrin (sf) proved to be highly sensitive and specific. this sf-elisa detected fibrin hacking fibrinopeptide a (fpa) via the monoclonal antibody 2t35 specific for the neoepitope generated on the aa-chain after the split of fpa. lill et al. recently introduced a new assay modification which utilizes the same antibody as the old one but takes advantage of a pretreatment of plasma specimens with kscn. this strong chaotropic ion is used to dissociate the various fibrin complexes possibly hiding fibrin epitopes. it was the aim of this study, therefore, to compare the two sf-elisa modifications (with and without kscn-pretreatment of specimens) . in order to examine the dynamics of thrombin-induced fibrin(ogen) metabolism we made course observations in patients with a certain form of septicemia. both assay modifications detected fibrin(ogen) derivatives which differed considerably in kinetics (n= 160 samples from 10 courses). the former sf-elisa (no kscn) correlated well with prothrombin fragments, thrombin-antithrombin !11 -complexes and with the release of fibrinopeptide a ( r > 0.96, n= 151). results of the new sf-elisa with kscn pretreatment of patients' plasma, however, correlated conspiciously well with d-dimer levels (r > 0.94) but distinctly less with the markers of thrombin generation (-0.12 < r < 0.29). this good correlation with d-dimer levels was unaccountable since the d-dimer maximum occured significantly later than the peak of markers of thrombin generation (p < 0.05). therefore, kscnpretreatment of fibrin specimens seems to lead to a change in the specificity of the fibrin assay despite usage of the same catching antibody. different half-iifes of differently composed fibrin complexes should be considered in trying to explain the findings. nevertheless, the results of the former assay without kscn-treatment correlated much better with the well-known dynamics of thrombin-induced fibrin generation during hemostasis activation than the data from the new assay modification. consequently, further examinations are necessary to specify the effect of kscn on soluble fibrin complexes and the resulting assay specificity. a rapid assay for the determination of the primary hemostasis potential (php) of whole blood has been developed (kundu et al, 1995) from the original method of kratzer and born. the new system employs a disposable test cartridge which holds the sample (citrated whole blood) and all components for the tests at the same time. the test procedure is very simple. the cartridge is loaded with -500 p.l citrated whole blood and is inserted into the platelet function analyzer (pfa 100aaw). the test is started automatically after a preincubation phase of 2.5 rain. the reaction starts with the contact of the whole blood and the capillary which is connected with a collagerdephinephrin coated membrane with a small aperture inside the test cartridge. under constant negative pressure the sample is aspirated and through the contact ofplatelets and vwf with collagen adherence and aggregation begins. the adhesion and aggregation process leads to the formation of a platelet plug which obstructs the flow through the aperture. the result of the php is reported as closure time (ct). additional parameters such as bleeding volumes are possible as well. first results show good reproducibility, normal values in the range of up to 150 sec. and a good discrimination of healthy donors from patients with congenital or acquired platelet dysfunctions. the system detects aspirin induced thrombocyte function defects and von willebrand disease. in ease of an abnormal result in the collagerdepinephrin system a second type of cartridge with a collagerdadp coating can be employed. in the majority of cases aspirin induced dysfunctions are normalized and could thus detect aspirin use. the proposed system may be a valuable tool for routine assessment of the primary hemostasis potential in a routine citrate blood sample laboratory. inducing mental stress in 20 young healthy male volunteers aged 20 to 40 ),ears with no previous history of thmmbophilia or a hemorrhagic diathesis was performed by a first time parachute descent from an altitude of 1000 meters. the purpose of this investigation was to find out whether there are any changes in the corpuscular and plasmatic fractions of peripheral blood. we were especially interested in elucidating changes in the procoagulatory and/or fibrinolysis systems. venous blood samples were obtained directly before and directly after the jump. flight time from the departure of the airplane to the landing of the parachutists was approximately 20 minutes. the maximum time that elapsed between the two blood withdrawals were 45 minutes. in a preliminary study with different voinnteem, certain fluid imbalances had been observed. absolute numbers of leukoeytes (6.9 vs. 9. l/n0, erythrocytes (4.6 vs. 5.1/pl), and platelets (246 vs.276/nl) significantly increased (p < .001), as well as the hemoglobin concentration from 145 to 156 g/l (p < .018). even though fluid imbalances before and after the jump had practically been excluded by measuring nearly identical hematoerit values (.41 vs..42), we noticed a marked drop in aptr (27 vs. 23 sec) and a significant increase in factor viii ~tivity. as a direct stress response, we found a rise in fibrinogen concentration (2.4 vs. 2.8 g/l) which is one of the shortest acting acute phase proteins. concerning reactive fibrinolysis, d-dimers showed an increase in concentration from 115 lag/l to still normal values of 192 lag/l, which was not significant due to low numbers of values (p = .086). we observed similar changes in fibrin monomers and prothrombin fragments fl+2. from other investigations on the kinetics of the activation of the procoagulatory system we know that maximum activil7 is not reached until 24 hours after initiation of activation.these investigations studied perioperative changes in different kind of operations which served as a control group concerning the degrees of tissue damage and resulting coagulation disturbances. to better understand these phenomena we plan to induce mental stress in a laboratoq' environment to further exclude unknow~a influences on the mechanisms which can activate the procoagulatory and fibrinolytic systems. triodena (t) 30/40/30 ug ee, 50/70/100 ug gestodene) were tested for their effect on hemostatic parameters. three groups (n=20) of healthy female volunteers were treated for 6 months with one of these oc. blood was taken before treatment (day 24-28 of pretreatment cycle, 0) and on days 18-22 of the 3 ~ (i) and 62 (ii) treatment cycle. indications of an activation of blood coagulation and fibrinolysis were detected as the plasma levels of prothrombin fragment f i+2 and of fibrin split product d-dimer and plasmin antiplasmin complexes were found elevated during treatment. the following main regulatory components of blood coagulation, activators and inhibitors, were investigated: factor vii antigen fviiag, fvii clotting activity fviie, circulating activated factor vii cfviia and antithrombin 3 at3 activity, total protein s antigen tps-ag, free protein s antigen fps-ag, protein s activity psact, circulating thrombomodulin etm fviiag, fviie and cfviia significantly increased during treatment; cfviia: 0: c 32.4 mu/ml a prethrombotic condition characterized by elevated levels of circulating soluble fibrin has been claimed to be a predisposing factor for accumulation of coronary thrombotic material in acute myocardial infarction. the present study includes 161 patients with clinical suspicion of myocardial infarction. blood samples were drawn by the primary care physician, upon arrival in the hospital, and after 2, 6, 12, and 24 hours of hospital stay. patients with myocardial infarction were identified by typical course in 12 lead ecg, and upon sequential determination of troponine t, myoglobin, ck, and ck-mb. patients with primary cpr were excluded from evaluation. soluble fibrin was measured by enzymun®-test fm (boehringer mannheim). patients with acute myocardial infarction display soluble fibrin levels within the normal range (< 5 ~tg/ml) during the initial two hours after onset of symptoms. there was no significant difference between patients with myocardial infarction and patients with coronary heart disease without myocardial infarction. slightly elevated levels were found in patients with atrial fibrillation, reflecting intracardiac fibrin formation. in patients without fibrinolytie treatment, a slight increase of soluble fibrin levels with a maximum after approximately 8 hours is observed. most patients with fibrinolytic treatment display a considerable increase in soluble fibrin, with maximum levels immediately after infusion of the fibrinolytic agent. four patients with pulmonary embolism showed soluble fibrin levels in the range of 40-300 [.tg/ml, which remained in the same range during the entire observation period. in conclusion, circulating soluble fibrin is not increased in patients with acute myocardial infarction and does not appear to be a predictor of acute coronary events. high levels of soluble fibrin in patients with fibrinolytic therapy may reflect release of fibrin from thrombotic material, but also de novo generation of fibrin due to release of active thrombin from thrombi not necessarily located in the coronary vessels. detection of elevated levels of soluble fibrin in patients with acute chest pain should result in careful examination for signs of pulmonary embolism or aortic aneurysm. the possibility to determine activated coagulation factors opens the question if data provide evidence of an activated coagulation or fibrinolysis and if this has a prospective value. we investigated patients with confirmed thrombosis, postsurgical septieaemia and also after liver transplantation. in all patients factor viia, xii, xiia and also the fibrinolytic parameters t-pa, pai-1, pap, plasminogen and a2-ap were determined. in addition, f1+2 and apc-resistance with heterocygote factor v-leiden-mutation and confirmed thrombosis. we found increased factor viia which showed partly also an increased fl+2. patients with other pathological results such as a reduced t-pa and/or increased pai-1 showed a low incidence of elevations in factor vii or f1+2. the activation of factor xii seems to be of minor importance in patients with thrombosis. a different picture is found in septic and transplanted patients. obviously factor xii-activation is of major importance in this group. a deterioration of the clinical symptoms is correlated with an increased factor xiia which is paralleled by a decrease of factor xiiactivity. the investigation of fibrinolysis parameters such as pai-1 and pap demonstrate a fibrinolytic disturbance of the balance. statistically significant are differences in septicaemic patients both in the surgical and in the internistical group in contrast to polytrauma patients. in patients with liver transplantations significant changes are apparently related to rejection of the transplanted organ together with a deterioration of the clinical picture. the possibility to detect activated coagulation factors may be a tool to detect changes in the hemostasis system at an early stage and to use this for an improved therapy. control of long-term oral anticoagulation is usually performed by serial determinations of the prothrombin time. however, the assessment of effective anticoagulation versus the potential risk of bleeding complication is difficult to achieve. molecular markers of blood coagulation activation might add valuable information in individual cases. we investigated 48 patients with thromboembolic manifestations (deep vein thrombosis n=22, pulmonary embolism n= 13, myocardial infarction n= 13) for one year beginning with admission to the hospital. tat, prothrombin fragments f 1 +2, d-dirner and fibrin monomer concentrations were analysed. all markers were significantly increased at the time of initiation of anticoagulant therapy thus reflecting a prethrombotic situation. patients suffering from venous thromboembolism demonstrated higher concentrations of tat and f 1 +2 in comparison to myocardial infarction (34.6 vs 12.3 pg/1, p=o.009; 2.8 vs 1.3 nmol/i, p=0.0025). f 1 +2, tat and d-dimer concentrations decreased gradually over the first 14 days of anticoagulant therapy reaching values within the established normal ranges in all cases. f 1 +2 and tat concentrations reflect the activity of the coagulation system during long-term anticoagulation whereas analysis of fibrin monomer yielded partly controversial results. we conclude that f 1 + 2 and tat appear to be superior to fibrin monomer for the individual control of oral anticoagulant therapy. the influence of thyroid failure on haemostasis is controversial. mainly hypoceagulable states have been described in clinically overt hypothyroidism. since hypothyroidism has been associated with an increased risk of atherosclerosis, we studied a wide range of haemostatic factors in untreated female patients with subclinical (b, n=42, age 59+13) or overt (c, n=8, age 55-zcj) hypothyroidism, as well as in hypothyroid women under 1"4 treatment (d, n=8, age 57+9) and euthyroid controls (a, n=80, age 50+14). simple screening tests (prothrombin time, activated partial thromboplastin time, fibdnogen), procoagulant factors (fvii, fviii, von willebrand factor), coagulation inhibitors (antithrombin ill, hepadn cofactor ii, protein c, protein s) and fibdnolytic factors (plasminogen, antiplasmin, plasminogen activator inhibitor, tissue plasminogen activator) were measured. results factor vii activity (vii:c), factor vii antigen (vii:ag) and their ratio were found increased in hypothyroid patients. factor viii activity showed the same tendency, whereas von willebrand factor ramained unchanged, as did all other parameters with exception of free protein s, which declined in overt hypothyroidism and in t4 treated subjects. these differences tended to diminish after exclusion of 26 women with estrogen replacement therapy for menopause, but the ratio vii:cnii:ag, as well as fvii:c still remained significantly higher in hypothyroid patients. conclusions: subclinical and overt hypothyroidism are associated with significantly higher levels of factor vii:c and vii:ag. the disproportionate increase in vii:c compared to vll:ag, as shown by their ratio, might reflect the presence of activated factor vii (vila), which in turn indicates a hypercoagulable state. this pattern becomes more pronounced with the concomitant estrogen replacement after menopause. exocytosis following platelet activation leads to translocation of cd62p (p-selectin), cd63, and thrombospondin, from cytoplasmic granules to the cell surface membrane, where these molecules, serving as activation markers, can be detected by flow cytometry. we here report detectability of these molecules preformedprior to platelet activation -inside the cytoplasm of resting platelets. two different methods are compared, i. e. using either methanol or the fix&perm kit (an der grub) for cell membrane permeabilization. in addition, interleukin(il)-ice is shown to be present in platelet cytoplasm after methanol treatment, but not after permeabilization using fix&perm. whenever cell surface positivity for a specific marker coincides with intracellular presence, blocking of the surface membrane sites prior to membrane permeabilization is required in order to obtain fluorescence intensity attributable to cytoplasmic staining. our data demonstrate the feasibility of the methods presented for the detection of intracellular platelet molecules. this technique should also provide a means for estimating the relative quantity of intracellular platelet antigens, provided the permeabilization procedure does not lead to antigen leakage or destruction. physical exercise activates the clotting as well as the fibrinolytic system as indicated in numerous investigations of exercise by running and by bicycle ergometer but not by swimming. the positive effect of an endurance training in coronary sport groups is induced also by influences on the hemostatic system. the influences are suppression of the clotting activation by the acute exercise and by an increased fibrinolysis response. different hemostatic parameters, therefore, were analyzed before and after swimming of male coronary patients (n=33; median ag~ 61 years, achieved heart rate: 68/min). indicating plasmatic clotting activation there was a significant increase in molecular markers tat and f1+2 among the coronary patients (tat from 2,1 to 3,4 pg/1; fi+2 from 0,92 to 1,1 nmol/1). the degree of clotting activation among the coronary patients was less than that observed in a group of young volunteers in a former investigation. this must be explained by existence of the coronary heart disease or by the higher age in the patient group. indicating an activation of fibrinolysis t-pa activity increased significantly in coronary patients (from 0,14 to 0,5 iu/ml) resulting in an unchanged balance between coagulation and fibrinolysis. from this findings of the hemostatic systems no increased risk of the coronary patients by swimming can be derived. a prerequisite, however, are precautions l±ke to devoid exercise in the anaerobic range, exclusion of major heart failure and of cardiac arrhythmias before begirming of the swim training. the principle of the fontan operation consists in anastomosing the right atrium to the pulmonary arteria, thus bypassing the right ventricle and using the only functional single ventricle as a pump for the systemic circulation. there are only few data about the influence of the changes in hemodynamics on coagulation and fibrinolysis. we investigated the coagulation system in 20 children and young adults aged 4 to 21 years in a general examination 4 to 61 months after fontan procedure. besides other abnormalities of the coagulation system, there were significantly increased values for the thrombin-antithrombin-iii-complex (tat) in 12 patients (60%). as a marker for an activation of the fibdnolytic system we found elevated plasmin-alpha2-antiplasmin-(pap-) levels in 14 patients (70%). less frequently, the concentrations for the prothrombin-fragments 1 and 2 (f1 and 2) (7 patients, 35%) or the d-dimer (2 patients, 10%) were increased. we didn't find significant differences in a clot-lysis-assay between fontanoperated patients and an age-matched control group. there was no significant correlation between activation of coagulation and clinical situation or diameter of the pulmonary arteria. whether the present data can help to estimate the risk for a thrombo-embolic complication following fontan procedure, still has to be investigated. the results of the clot-lysis-assay suggest, that for lysis of thrombi the same dose of rt-pa should be used as for other patients. a 2nd generation functional protein s assay p. van dreden* and e. adema** * serbio, gennevilliers france, ** boehringer mannheim, tutzing germany a second generation protein s test was developed with improved sensitivity to protein s and better reagent stability. the test result was found to be unaffected by apc-resistence (10 patients, heterozygote for the mutation with a apti' + apc ratio between 1.4 and 1.9), heparin up to 2 iu/ml and f viii activity between 1 and 250%. in the test, diluted sample is mixed with protein s deficient plasma, activated factor v, activated protein c, phospholipids and an intrinsic pathway activator. this mixture is incubated for 3 minutes. during this time, the activated protein c inactivates part of the f va. the extend of f va inactivation depends on the protein s concentration. after 3 minutes caci2 is added and the time untill clot formation is measured. the clotting time is a linear function of the protein s concentration between 10 and 140% protein s. for the three preproduction lots the difference in dotting time between 10 and 100% protein s was 43-54 seconds. this compares to 30-40 seconds typically obtained with the old test. within run precision (n= i0 on sta) is cv= 2 -7% on the basis of protein s. day to day precision (n=10 on sta) was found to be cv= 4 -11%, again calculated on the basis of protein s concentration. the cv of 11% was obtained for an avk plasma with 13% protein s; it corresponds to a standard deviation of only 1.5% in protein s. the insensitivity to interferences, in particular apc-resistence and better precision and stability are expected to improve the quality/reliability of a protein s determination. in this study we evaluated the use of hormonal contraception on the parameters protein c, protein s and pal. samples from 71 women with, without hormonal contraception and in menopause were assayed by coagulometric (protein s clotting test (behdngwerke, marburg, frg) or chromogenic methods (protein c activity test and pal reagent from behringwerke, marburg, frg) in double determination and were compared with the reference ranges. in addition thromboplastin time (thromborel s reagent) and fibrinogen (multifibrin) from behringwerke, marburg, frg, and aptt (actin fs reagent from dade corp., unterschlei6heim, frg) were determined. in women using hormonal contraceptives (p<0,01) and in menopause (p<0,05) protein s activity was significantly reduced compared to other women (<45 years) while protein c acitivity did not change. in menopausal women a higher susceptibility to thrombosis was supported by an increase of aptt (p<0,05) and fibronogen (p<0,01). while there was no change for pal, plasminogen was significantly lower in women using hormonal contraceptives and in menopause (p<0,05). we could not observe a higher turnover of coagulation and fibdnolytjc system with hormonal contraception. noteworthy was the occurence of low (<200 mg/dl) and borderline fibrinogen (max. 220 mg/dl) in 40,9% of women res. in 22,8% of women (together with borderline aptt) who had an individuell risk for arterial disease. protein s protein c fibdno~en aptt plasminog~ without hcc 109,1-+13,6 78,3-+14,1 255,0-+14,0 36, [2] [3] [4] [5] 4 24, [0] [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] 2 with hcc 85, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] 2 78, 5±12, 0 253, 8.+24, 1 35, [2] [3] [4] 8 14, 9 menopause 90, 3+97, 6 87, 4±41, 4 307, 05:57, 6 39, [8] [9] [10] 0 12, 6 hcc= hormonal contraception hemostatic parameters in a patient undergoing bone marrow and subsequent liver transplantation due to veno-occlusive disease c. salat 1, , e. holler t,3, hi. kolbl, 3, b. reinhardt l, r. pihusch 1, p. g0hring 2, s. poley 2, e. hiller 1 l=med. klinik iii, 2 = institut flit klin. chemie, klinikum grosshadern der ludwig-maximilians-universit~tt mfinchen, 3=h~tmatologikum der gsf a 40 year old patient suffering from all received allogeneic bone marrow transplantation (bmt). after an uncomplicated early posttransplant period the patient was dismissed after 4 weeks. a bilirubin rise with subsequent liver failure was observed during the following weeks. according to biopsy proven hepatic veno-occlusive disease (vod) liver transplantation was performed on day 79. unfortunately the patient died on day 140 due to aspergillosis. we monitored levels of protein c (pc) and s (ps) as well as pall during the pre-and posttranspiant period. pal1 level was normal (<43 ng/ml) during the first 4 weeks after bmt but increased with the manifestation of vod (317.5 ng/ml on day 47). it reached its peak immediately before liver transplantation (547.6 ng/ml) and returned to normal levels within the next few days. pc levels which were normal before bmt decreased prior to clinical diagnosis of vod and were normal after liver transplantation. ps levels lay within the normal range at all timepoints. vwf was elevated before bmt (240%) and remained relatively stable during the whole investigatonal period ranging from 170 to 260%. it is assumed that vod is initiated by an endothelial cell injury -possibly due to radiochemotherapy -and subsequent hypercoagulability. our results indicate that the "endothelial cell marker" vwf is not helpful in predicting vod. the kinetics of the investigated parameters underline the significance of pc and pai-1 as described by others and our group earlier, whereas ps does not seem to play a role in the pathogenesis of vod. the budd-chiari syndrome (bcs) is characterized by hepatic venous outflow obstruction that may be caused by the precipitation of a thrombus. it frequently coseggregates with other major diseases like myoloproliferative diseases or defects in the haemostatic system (antiprotein c and protein s deficiencies e.g.). only recently, the factor v leiden mutation (fvlm) has also been associated with bcs. we hypothesized that defects in the thrombo-modelling associated anticoagulant pathways (tmaap) are a major risk factor for the precipitation of bcs. we screened our cohort of 27 patients (pts) with bcs for the presence of defects in the tmaap and identified 3 pts with protein s deficiency (psd). these pts were screened for the three point mutations in exon 1 (codon-25; ins t), exon 15 (codon 636; a-->t) and in intron 10 (g-->a + 5) of the ps alpha-gene that have been demonstrated by bertina et al to coseggregate with psd. restriction enzyme analysis and confirmation-sensitive gel electrophoresis for the detection of single-base differences in doublestranded pcr-products were employed. all living family members of the indicator pts were also screened for heterogeneties in the three point mutation as described. no single abnormality in these genes despite presence of pbd in those family members was found. in addition, pts and family members were also screened for fvlm. one pt and two of his family members, in addition to psd, were subject to fvlm. the other two lots and their family members were not subject to fvlm. in contrast to the first family, despite psd, those two pts suffered from morbus crohn and acute myeloid leukaemia as risk factors for bcs. we conclude: psd is one major risk factor for the precipitation of bcs. to precipitate this disease, one additional risk factor is required. psd may be caused by genomic defects in the protein s gene other than those described by bertina. only a few publications describe a thromboembolic disease due to dramatically reduced protein s levels being associated with viral or bacterial infections, autoimmune mechanisms are suspected but the aetiopathogenesis is still under discussion. we report on a 5 year old boy who developed purpura fulminans of the left leg during varicella infection. on the fourth day of infection the disease started with pain and haemorrhagic efflorescence localized at the left taft. on admission the boy suffered from a purpura fulminans with central necrosis measuring 15x8 era. suspecting a hereditary thrombophilic disease we started therapy with protein c concentrate and recombinant tissue type plasminogen activator. the fellowing coagulation investigation showed a severe deficiency of protein s (total protein s-antigen < 5 u/ml, free antigen not measurable) in combination with factor v leiden mutation. other thrombophilie and coagulation parameters did not show deviation from normal range. after 4 weeks we saw a slight improvement of the total protein s antigen up to 50 u/ml. the free protein s antigen was still undetectable. during the following weeks the patient recovered slowly and the protein s activity and antigen normalized. because of skin necrosis thromboembolie prophylaxis was initiated with low molecular weight heparin (fragmin®, 100 ie/kgbw/die) and continued for 6 months. under this therapy there were no further thromboembolic events. these results suggested an autoimmune protein s deficiency in a patient suffering from chickenpo×. an analyses of autoantibodies at the time of diagnosis showed a slight increase of the antieardiolipin antibodies (igg 16,1 iu/ml, igm 15,1 iu/ml) which normalized during hospitalisation. we suspect an antibody to protein s probably caused by similar presented viral antigens. we suppose that autoimmune mechanism during different infections in combination with a heterzygous apc-resistance may be a potential risk factor for developing thrombotic disease. in the central nervous system mrna encoding for prothrombin and thrombin receptor is present and astroglial cells in culture process and secrete thrombin. moreover, effects of thrombin on brain cells including change of neudte outgrowth and astrocyte shape are described, but the molecular mechanisms are unclear. we investigated the effects of human <z-thrombin and a six amino acid thrombin receptor activating peptide (trap-6, sfllrn) on [ca2+]i, phosphoinositide hydrolysis and protein kinase c activation in the rat glioma c 6 cell line which is a widely used in vitro model for studying glial functions. stimulation of c 6 cells with both c{-thrombin and trap-6 resulted in transient [ca2+]i mobilization, [3h]inositol phosphate response and activation of the pkc isoenzymes c{,i],7,5, and s. these results suggest that ~-thrombin and trap-6 activate at least partially the same intraceltular signaling pathways in rat glioma c 6 cells which is an evidence for involvement of "tethered ligand" receptor in thrombin induced signaling in glioma c 6 cells. further investigations in this field are in progress including both receptor binding studies and more detailed analysis of thrombin receptor mediated signaling network and the cellular response of thrombin induced transmembranal signal transduction pathways in c 6 glioma. thrombin is a potent mitogen in vascular smooth muscle cells (smc). the mitogenic effect of bovine thrombin and human thrombin receptor-activating peptide (hutrap-14) was investigated in bovine coronary artery smooth muscle cells. confluent cells (passage 4-8) were incubated for24 h in serum-free medium with thrombin (10 nm) or trap (10-100 pm). the mitogenic response was assessed using [sh]thymidine incorporation. results demonstrated that thrombin induced a three-to fourfold increase in [3h]thymidine incorporation compared to the control, thus being as active as the potent mitogen pdgf-bb (20 ng/ml). the mitogenic effect of thrombin was dependent on its proteolytic activity. when the active site of thrombin was irreversibly inhibited by d-phe-pro-arg-chloromethylketone (ppack) no increase in [°h]thymidine incorporation was observed even at the high concentration of 250 nm ppackthrombin. thrombin-induced [3h]thymidine incorporation was significantly reduced by the reversible direct thrombin inhibitor nc£-(2naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide (napap, lpm) . in contrast to thrombin trap-14 at a concentration up to 100 pm did not increase [3h]thymidine incorporation in the absence or presence of the aminopeptidase inhibitor amastatin (10 ijm). in order to demonstrate whether hutrap caused a stimulation of the receptor and signal transduction in bovine smc the activation of protein kinase c (pkc) was measured as translocation of the cytosolic to the membrane bound fraction. it was found that both thrombin (10 nm) and trap (10 pm) induced a translocation of pkc after 20 rain. in conclusion, thrembin and trap activated pkc in coronary smooth muscle cells but only thrombin acted as a mitogen. endothelial cells once stimulated with thrombin are resistant to subsequent stimulation directly after the first. but after a recovery period of about 90 min the cells are sensitised again for activation by thrombin. in our experiments this resensitisation couldn't be inhibited by actinomycin d or cycloheximide nor by the phosphatase inhibitor ocadaic acid. therefore an intracellularly pool of thrombin receptors has been proposed possibly co-localised with golgi vesicles. brefeldin a (bfa) has been used extensively to investigate the intracellular sorting of proteins because of its dramatic alteration of the structural and functional organisation of the golgi apparatus. because of this we examined the effects of bfa on the regeneration of the thrombin receptor response in human umbilical vein and artery cells. the vwf release from weibei-patade vesicles was used as physiological parameter of activation of thrombin receptors by thrombin. the addition of bfa (20 pg/ml -20 ng/ml) to endothelial cells blocked the receptor response regeneration, whereas the first stimulation was not influenced by bfa at the concentrations used. these results give further evidence that the proposed storage pool of thrombin receptors is iocalised in vesicles of the golgi apparatus. randomized comparison of double bolus reteplase (r-pa) and front-loaded alteplase (rt-pa) in patients with acute myocardial infarction (rapid ii) c. bode, rw. smalling, j. kalbfleisch, g. berg, dj. odenheimer, e. bshm, wd weaver and the rapid ii investigators. med. klinik iii, university of heidelberg, germany the study was undertaken to assess the efficacy of a double bolus regimen of 10 + 10 megaunits of reteplase, a recently developed deletion mutant of tissue-type plasminogen activator, relative to front-loaded (gusto-regimen) alteplase. there was no age limit and patients were recruited up to 12 hrs after onset of symptoms. the primary endpoint "patency at 90 minutes" was centrally assessed in a blinded fashion. infarct related coronary artery patency (timi grade 2 or 3) and complete patency (timi grade 3) at 90 min for reteplase vs. alteplase were 83.4% vs. 73.3% (p=0.03) and 59.9% vs. 45.2% (p=0.01), respectively. at 60 minutes, the incidence of both, patency and complete patency was also significantly higher in retep ase vs. aiteplase treated patients: 81.8% vs. 66.1% (p=0.03). and 51.2% vs. 37.4% (p=0.01). reteplase treated patients required fewer additional coronary interventions within the first 6 hrs of treatment (13.3% vs. 26.5%, p=0.01). there were no significant differences between reteplase vs. alteplase regimens in 35 day mortality (4.1% vs. 8.4%), severe bleedings (12.4% vs 9.7%) • or hemorrhagic stroke (1.2% vs. 1.8%). reteplase, when given as a double bolus of 10+10 mu to patients with acute myocardial infarction, achieves significantly higher rates of early patency and requires fewer acute coronary interventions than front-loaded alteplase without an apparent increased risk of complications. lysis therapy with uhsk of deep vein thrombosis (dvt) of the lower extremity commoniy results in a relatively high patency rate. in our patients, patency was achieved in approx. 70-80%. these results were supported by several studies. in dvt of the pelvic veins, however, this kind of aggressive therapy is excluded due to the risk of iatrogenically induced pulmonary embolism. on the other hand, the need for an effective therapy is evident because of the usually occtnring complete occlusion of the pelvic veins and the following severe clinical symptoms 0phlegnmsia cerulca dolens etc.). until september "95, 44 consecutive patients (16 males, mean age 41 ranging from 18 to 63 years) with dvt of pelvic veins were included in this study. after clinical and phiebographic/computer tomographic (ct) diagnosis, a temporary filter system was placed intravenously either by a wambmchial (34 pat.) access route, lransfemorally (6 pat_) or wansjngularly (4 pat.) under mdiological control. the open filter was positioned above the thrembus in the lower caval vein: 26 times an antbeor system, 11 times an anglocor and 10 times a cook filter were used. in the following three days up to 3 cycles of lysis therapy with 9 mli u of streptokinase were administered, each cycle lasted six hours. heparin was given simultanously via the filter system in therapeutic dosages in order to prolong alrit values 1.5 to 2 times the individual base line level. effective lysis was controlled by a second phlebography/ct, and the filter system removed through the introducer sheath valve. after lysis therapy all patients were put on coumarin with overlapping heparinization. successful lysis (complete recaanlisation without remaining thrombi and lysis of more than 50 per cent of the original thrombus) could be achieved in 62% (29 patients). in two cases a fatal pulmonary embolism occurred. 5 times great parts of a thrombus were captured by the filter and could be removed with the system. due to an insufficient boparin therapy we detected a newly developed thrombosis of the lower cavul vein twice after lysis. this complication could be resolved by additional administration of streptokinase. in nearly all cases we found extensive hematomas on the catheter insertion sites. 4 patients with transfemoral applications had to be transfitsed with red packed cells due to retroperitoneal hematoma. one filter system was broken after use. lysis of dvt of the pelvic veins with uhsk and with the protection of a removable temporary filter system situated in the lower caval vein enables us to carry out an effective therapy. the filter systems, however, have to be ameliorated in order to better prevent fatal pulmonary embolism. heparin should be administered at least in the remainder of the day of lysis. the insertion site should be the enbital vein because compression for bleeding complication is easily feasible at this location. we report about seven of our patients (pts.) with bcs treated by fibrinolytic therapy (5 female, 2 male, age 27, 6 years + 8). only one case was of unknown origin while in four pts. myeloproliferative syndromes, in one protein c deficiency and in another one morbus behcet was detected in five pts. chronical and in two acute bcs was diagnosed. as complications were found: in three pts• thrombosis of vena iv.) cava, in one ofv. porta and v. lienalis, in one more of v• porta and v• cava. we treated just one patient with urokinase (3 x 800.000 iu/d for 18 days). four pts. were treated with rt-pa in concentrations of 25 to 50 mg/d (for a period of up to 14 days) and continuous heparin infusion. high doses of rt-pa (80 to 120 mg/d for one or four days) were given in one case of acute and one of chronic bcs. partial (after high-dose rt-pa nearly complete) recanalisation of thrombotic hepaticel veins was only achieved in the two acute cases of bcs. no significant recanalisation was proven in hepatical veins of chronic bcs. three times complete fibrinolysis of v. cava thrombosis (two after highdose therapy) and one partial lysis could be attained. in one patient partial lysis of v. porta and in another one of v. lienalis was found. furthermore complete recanatisation of v. porta followed local rt-pa lysis during surgical intervention. in our two pts. with acute bcs significant improvement of hepatical venous flow could be measured after fibrinolytic therapy• in chronic bcs hepatical venous flow did not improve while the complication of v. cava thrombosis could eventually be treated successfully. probably due to fibrinolytic therapy one case of esophageal varix bleeding occurred. the benefit of flbrinolytic therapy of bcs seems to be influenced by the dur~ion of the disease• according to our results fibrinolytic therapy should be considered in cases of acute bcs as well as in the above mentioned thrombotic complications• recent studies suggest that transport of thrombolytic agents through the clot can play a major role in determining the reperfusion time needed for thrombolysis. as the mechanisms of this phenomena are rather unclear we studied fibrin (0.4-1.5 ~tm) clot lysis by plasmin (30 nm) presented outside or inside the clot in purified system (tris-hc1 buffer, 37°c). the clots was formed by addition of cac12 (30 mm), thromboplastin (1 u/ml) and human thrombin (6 nih/ml)t the lysis degree was determined either by counting of radiolabelled fibrin degradation products or by time of total lysis. obtained kinetic parameters of this reaction: kest=99.4 rain -1, km=i.9 ~tm (plasmin inside clot); kcat=l.36 rain -1, km=i.3 lzm (plasmin outside clot). this data indicate that eatalytie efficiency of plasmin from inside the clot is 50-fold higher than from outside as measured from kcat/km. furthermore we studied plasma clot (0.25 ml) lysis in original plasma (3 ml) containing sk (5-100 nm) and seu-pa (5-100 rim) in the final concentrations from inside or outside the clot. the results are presented in the the similar results have been got when the fibrin clots were formed with batroxobin instead of thrombin. it may be concluded that fibfinolysis is a surface-dependent process. it's likely that the higher level of plasminogen activators in blood would prevent thrombus formation, while physiological clots would stay resistant. for the improvement of the thrombolytic therapy in children more clinical data are needed. therefore we report on our experiences with rt-pa presenting the clinical course in 11 patients (age range: 7 days to 16 years) with venous thrombosis (n=9) and purpura fulminans by meningococcosis (n=2). the patients were treated with rt-pa (actilyse r) for 10,5 hours to 13 days. the venous thromboses were localized in lilac/femoral veins (n=4), brachiocephalic/jugular/subclavian veins (n=3) and the cava superior vein (n=l). central venous catheters were causative in 2 cases. the dosage range was between 0.2 to 0.5 mg/kg for the initial dose and 0.8 to 2.0 mg/kg/d for the subsequent continous infusion. ten patients received simultaneously heparin. complete clot dissolution occured in 6 cases, partial clot dissolution in 1 patient. in 4 patients with a history of the first clinical signs of thrombosis of 3 weeks or more the lysis was not successful. concerning the side effects only small bleeding episodes on former venipuncture sites were observed in 5 patients. systemic effects such as the decrease of the plasma flbrinogen concentration were rare too. in conclusion, the thrombelysis with rt-pa presents an effective and safe therapy in children at: the rt-pa dosage used. in case of a history of the thrombotic event of more than 3 weeks a thrombotytic therapy should not be carried out. d.dimer is widely used in clinical situations associated with thrombotic diseases. the most popular application is the exclusion diagnosis of deep-veinthrombosis and pulmonary-embolism and the assay is performed in emergency. d.dimer is also frequently measured for evaluating the thrombotic risk in the post-operative period. quantitative, flexible and sensitive assays are required. we have developed a new automatic method which works on the coagulation walk-away instrument, sta. this assay was developed with two monocloual antibodies specific for d.dimer coated on microlatex particles and it uses an immuno-turbidimetric technology. absorbance is measured at 540 nm and is a direct relationship of d.dimer concentrations. the assay works with 50 ~tl undiluted plasma and is performed in less than 10 minutes. the dynamic range is from 0.2 to 16 [~g/ml and no prozone effect is observed up to concentrations higher than 100 ~tg/ml. the detection threshold is of 0.2 [lg/ml. the intra-assay reproducibility is below 5% and the inter-assay reproducibility is below 6%. recovery studies of purified d.dimer added to normal plasma were between 94 and 105%. no interference of heparin, bflirubine, lipids or of rheumatoid factor up to concentrations of 100 u/ml is observed. there is no effect of high fibrinogen concentrations (> 10 g/l). when compared with conventional elisa techniques (asserachrom ddi), the assay demonstrated a correlation coefficient of 0.97 on 131 samples from normal individuals and hospitalised patients with elevated d.dimer concentrations. slope was of 0.97 and intercept was of -0.07. this new assay offers a full flexibility for individual testing as the calibration curve is stable for at least one week on the instrument. it is then well adapted for all the applications of d.dimer measurements in coagulation laboratories. 16 children between an age of 3 days and 11 months ( median 6 weeks ) with thrombotic or embolic occlusion of major vessels were treated with rt-pa for thrombolysis. the affected vessels were both sided renal veins or one sided renal vein and v. cava inf. in 8 cases, the v. cava superior in 3, the v. cava inf. plus renal veins plus aorta in 1, the left ventdcle in 1, the aorta in 1, the a. femoralis in 1 and the v. portae in 1 case. 10 out of 16 occlusions were associated with an indwelling catheter. underlying dieseases were sepsis (4), prematudty (3), vitiurn (2), asphyxia (1), short bowel syndrome (1), hus (1), diabetes (1), cmv (1), exsiccosis (1) and m. hirschsprung (1). thrombolysis was performed with an bolus of rt-pa (0.1-0.2 mg/kg) followed by continuous infusion (0.8-2.4 (-9) mg/kg/24h, median 1.8 mg/kg/24h). low dose hepadn (100 ie/kg/24h) was given dudng full dose hepadn (aptt 1,5-2 times normal) after the thrombolysis. in 5 pts. rt-pa was administered locally through the catheter and in 11 cases systemically. in 13 patients the vessels could be recanalised completely, in 2 partially, in 1 patient the therapy had to be discontinued. in 2 vessels a reocclusion occurred. bleedings were noted in three patients, all from recent venous puncture sites. the results encouraged us to start a multi-canter trial which has been approved by the ethical committee and is open for recrural. the aim is to compare efficacy and safety of rt-pa with urokinase, the only recommended standard in the management of critical major vessel obstruction in newborns and infants. the design is a randomised, notblinded trial with a cross-over option after three days in cases without success. study end points are recanalisations, major bleedings and number of cross-overs. inclusion criteria are age under 1 year, lifethreatening vessel obstruction, age of thrombus up to 10 days, no precaeding fibdnolytic therapy. exclusion cdteda are cerebral hemorrage, pedventricular leukomalacia, surgery dudng the last 7 days and cns injuries during the last 2 months. although our knowledge on inherited thrombotic coagulation disorders has greatly expanded within the last years, there are still man}, patients with recurrent venous thrombosis in whom no obvious predasposition can be identified.thus we decided to include also so-called rare defects associated with thrombosis in our routine thrombophilia screening programme, such as fxii deficiency. fxii is an important element m the intrinsic pathway of fibrinolysis and there is evidence for an insufficient fibrinolytic activity in fxii deficient pts..up to date only few and controversial data exist about the frequency of fxii deficiency in pts. with thrombophilia. cons~uently the aim of our study was to evaluate the association between fxii deficiency and juvenile venous thrombosis in a great population. patients and methods: 1554 pts. (851 female, 703 male, aged i to 61 ys, median age 38.2 ys) with venous thromboembolism before the age of 45 ys were studied. one-stage clotting activity assay of fxii (fxii:c) was performed on acl using fxii deficient plasma from instrumentation laborato~. fxii antigen concentration (fxii:ag) was measured by electroimmundiffusion using reagents from behfingwerke, enzym research respectively. the normal ranges are tl~. routine reference values obtained m our labratory from 80 healthy subjects (40 males, 40 females, median age 26.2 ys); 95% range: fxii:c 53-135%, fxii:ag 57-132%). results: 122/1554 pts.were classified as fxi1 deficient (f 60, m 62), giving a prevalence of 7.8%. severe fxii deficiencies with fxii:c below 1% were observed in 7 pts..ll5 pts= proved to have moderate fxii deficiency with fxihc ranging lrom 2 to 51% and fxii:ag ranging from 1 to 53%. in none of them inherited deficiencies of other well established thrombophila risk factors could be detected. none of the fxii deficient pts. had positive lupus anticoagulant tests. familial fxii deficiency was found m 9 cases. discussion and conclusion: the precedences of fxii deficiency amongpts, with venous thromboembolism was previously described to be 7.5-10%. supporting these data, we have shown a praevalence of fxii deficiency of 7.8 %. in comparison to the frequency of other well established thrombophila risk factors we consequently have observed a relatively high prevalence of fxii deficiency m our study group.these data, from the largest such study reported, strongly indicate that fxii deficiency may not be a rare deficiency and may be more frequently associated with thrombosis than currently suspected. we describe a family with an exceptionally rare, i.e. plasminogen, deficiency, combined with subnormal activities of coagulation factor xii (hageman factor). the first thromboembolic event, pulmonary embolism in the proposita was diagnosed at age 35. since that time, 'spontaneous' venous thromboembolic events verified by phlebography and perfusion/ventilation lung scan recurred once every year despite oral coumarin therapy, whose intensity varied over an exceptionally wide range despite tight control the patient was repeatedly given succesful thrombolytic therapy with streptokinase or recombinant tissue plasminogen activator. her plasma plasminogen chromogenic activity was 51-59 % compared to a normal plasma pool (reference range 70-130 %), plasminogen antigen was diminished to the same extent. the patient's factor xii exhibited only 28-55 % activity in a factor-deficient plasma assay as compared to a normal plasma pool. other known risk factors for recurrent venous thromboembolism were not present : no evidence of malignancy, no obvious precipitating events, normal values of antithrombin iii, protein c, protein s, fihrinogen, thrombin time, platelets, lupus-like anticoagulant, aptt prolongation after addition of activated protein c. the proposita's mother had died at age 60 from pulmonary embolisnt no coagulation studies are available. the proposita's sister was first diagnosed deep leg vein thrombosis at age 17, since that time recurrent episodes of venous thromboembolism have been diagnosed also in an other hospital. this sister's plasminogen activity was 120%, but factor xii activity was reduced to 55 %. three brothers of the proposita were examined, too, all in their 3rd decade of life. none of them recalled symptoms of or treatment for thromboembolic disease. in one brother, factor xii activity was normal (100-105 %), but plasminogen only about 50 %. in the 2nd brother, factor xii was very variable (64, 42 and 94 %), plasminogen was in the lower normal range, in the 3rd brother, factor xii was about 50 % (repeatedly), plasminogen was normal. current knowledge about the risk of thromboembolism with both enzymes is limited, the optimal management remains controversial. msrgit serbsn,maria cucuruz,dan madras,carmen petrescu, natalie rosiu,rodica costa iii rd psediatric clintc,universtt v of medicine, the unsatisfactory efficiency of entihepetitis b vaccination in our haemsphiliscs suggested the control of the immune status in 52 hiv negative patients,by establishing through flowcitomstrie with monoclonsl antibodies the lymphocyte subsets (cd3,cd4,cds,cd&/cd8 ratio and cd19) and by seric tmmunoglobulins levels; the immunological parameters have been correlated with the serological markers of hepatitis infections (hay, hbv,hcv ebd hdv) as well as on dependence with the treatment (blood,plasma,crysprecipitate,fector viii/ix concentrate) and the quantity of their consumption (ui/k9 weight/yesr).the interpretation of the results pointed out • significant lower level of cd3,cd& (p<o,o01) st the group of hsemophiliscs treated with blood,plasma and cryopraclpitate; sdditionsly,in this group of patients it was found s significant drop of the cd19 counts (p< 0,001) without an immunoglobulin-deficiency. the same behaviour was found at the group of haemophiliacs carriers of hb~ and hcv markers.the superpositisn of hepatitis infections on the treatment with blood and native blood products in most of the cases has let unanswered the question related to the mechanism of the immunodeficiency: the hepatitis b or c infections or the negative impact of the itarative important alloantigenlc load ? illumination with l~tm methylenblue (mb) is used for inactivation of enveloped viruses in human plasma. via a photooxidative mechanism viral structures as well as plasmaproteins especially fibrinogen can be damaged. fibrinogen is an important mediator in the cell-cell interaction of platelets. the four rgd sequences (arg-gly-asp) of fibrinogen can be recognized by the activated fibrinogenreceptor gphb/iiia . after binding to gpiib/iiia one molecule fibdnogen can link two platelets. the aim of this study was to investigate the influence of mb treatment on the fibrinogen activity in human plasma. furthermore we were interested to demonstrate if altered fibrinogen inhibit platelet aggregation by blocking the gpiib/]iia receptor. human plasma was treated with 1-50~m mb. after illumination fibdnogen was isolated by fl-alanin precipitation. purified fibdnogen was analysed by one-and twodimensional gelelectrophoresis. influence of modified fibrinogen on platetet aggregation was measured with gelfiltrated platelets. possible receptorblockade were examined in an 125j-fibrinogen competition assay. densitometric scanning of reduced sds-pages showed a decrease of the a-subunit as well as an increase of high molecular aggregates with increased mb concentrations. this effect was not detectable for fibrinogen isolated from plasma with 1bm mb. this results were confirmed by two-dimensional gelelectrophoresis. ibm mb treatment showed no receptor blockade in aggrement with normal platelet aggregation after stimulation with adp and fibrinogen. proposed validation of a quantitive quality-assured pcr method f. dorner, j. eibl, g. zerlauth immuno ag we have developed a highly sensitive and reliable quantitative method, based on the polymerase chain reaction (pcr) and on the reverse transcriptase polymerase chain reaction (rt-pcr), to screen for the nucleic acids of human immunodeficiency virus type 1 (hiv-i), hepatitis b virus (hbv) and hepatitis c virus (hcv) in plasma and plasma products. the detection of pcr products is carried out by laser induced fluorescence, the procedure is therefore termed "laser induced fluorescence pcr" (lif-pcr). the procedure allows the precise quantitation of genome equivalents due to the use of two calibrators that are co-amplified by the same set of primers as the target template in one and the same test tube. a high degree of reliability is achieved by co-precipitation of the extract, co-amplification and co-determination of the calibrators together with the genome equivalents to be determined. in this report we present validation data for the lif-pcr and discuss them in the light of the recently published ich-guidelines on the validation of analytical procedures. taking into account the pecularitles of the replacement therapy of haemcphiliacs in romania the study aimed at evaluating the dimension and profile of blood-born infections in these patients; 142 hsemophlllacs,elasslfled in 3 groups: a-ulth no exposure to blood or bloodproducts, b-treated ~ith factor-concentrates and c-ulth plasma,cryoprecipitate + factor-concentrates in their therapy have been investigated (elisa method) for serological markers of hepatitis a,b,c end d, cvtomegalovirus (cmv) and hiv i and hiu 9 infection. the infections profile of our haemophiliacs~hes some peculsrities. the hepatitis infection is the most extended,lnvolving more than 75% of the patients. the frequency is for hepatitis -bz,z~ , u -"/~,~ , g -bb,7% end o -24% : the multiple infection was diagnosed in more than 50% of cases. the prevalence uas significantly hlgher in the group c. we studied blood count and relevant coagulation system parameters of autologous blood donors. indications for autologous blood donation were divided in 4 categories: a group of diverse (a, n=13), reduction mammoplasty (b, n:5), prostatectomy (c, n=io) and total hip replacement (d, n=74). we performed bleod count (bc), thromboplastin time (tt), partial thromboplastin time (aptt), fibrinogen (fib), f vhi act, vwf rco, vwf ag, f xiii, at iii, d-dimer-fragments (dd) and fibrinmonomers (fm). all parameters were analyzed as initial value, bc, tt, aptt, fib, f xiii and at iii were measured additionally at each donation. the percentage of initial pathologic data was (1) were included in the study. the inclusion criterittm was: an endegen(y.~ red cell reserve (ercr) of less than 400 ml in men and less than 540 ml in women. ercr is the volume of blood that can be withdrawn up to tim hematocrit of 0.34. the selected dosages of rhepo (200, 400 and 800 iu/kg body weight biweekly over 4 weeks) were based on the calculated blood volume and the ercr. a unit of whole blood, separated into an autologens red cell concentrate and one unit of fresh frozen plasma, was collected when ]mnoglobin concentration was at least 11.5g/100 ml (or hematocrit was at least 0.34) up to an autologons predeposit of 800 ml red cell volume (rcv). additionaly, platelet concentrations and serum ferritin levels were measured~ results: at the initial clinical examination the ercr was 333 ml in women and 307 in men. this resulted in a weekly dosage of 2x32.000 iu rlaepo in women and 2x25.000 iu rhepo in men. 7 patients donated 5 red cell each (858 ml rcv on an average) within 19.1 days. the targeted rcv could not be reached in 3 patients because the operation was reschednled. the collection from one patient had to be aborted because of angina pcetoris complains. tic following side-cffczts occurred: episodic hypcrtejlsion (2 tim~), chest pain (5), fafigne (8), inflnenza-lihe syndrome (3). platelet count increased from initially 203 4-68 x 10'//d (i00 %) during therapy to a maximum level of 260 + 93 10v~ (128 %) without any clinical signs of thrombeembolism. in the control group (autologous blood donation weekly 3 or 4 times without erythrupoiedn stimulation, n=12) platelets increased from initially 230 __+ 73 10'/fi (100%) to 255 + 71 10v#i (110%) i1]aximlzm colldttsions: the adlninist/'ation of lhepo in the above described manner to autologous donors undergoing heart surgery is well tolerated. it allows to collect a sufficient number of autologous blood units and the preparation of a predeposit at short notice. essential thrombocythemia is a chronic myeloproliferative disease with a benign clinical course that is mainly complicated by microcirculatory or thrombotic vascular disorders. it is at present uncertain which patients need (lifelong) therapy and whether cytoreduction is necessary. we retrospectively evaluated vascular complications in 128 patients (33 men, 95 women) with a median age of 61 years in relation to therapy. three major treatment groups were analysed: 17 patients treated with acetylsalicylic acid (ass) alone, 29 patients given alkylating agents, and 62 patients treated with interferon alpha (ifn). there was no significant number of patients treated with hydroxyurea. ifn-treated patients were younger (median age 54 years) than patients in the other groups, but platelet counts before therapy were similar (median 0.84-1.13 x 1012/i). before therapy, 47% of patients treated with ass, 45% treated with alkylating drugs, and 32% treated with ifn had bleeding events, microcirculatory or thrombotic complications. dudng therapy, median platelet count was 0.9 x 1012/i in patients on ass, 0.68 x 1012/i in patients treated with alkylants, and 0.48 x 1012/i in ifn-teated patients. the rate of patients with complications during therapy was 18% (9.5%/patient year) for ass, 14% (6.3%/patient year) for patients on alkylating drugs, and 15% (6.2°/dpatient year) for ifn-treated patients. we conclude that all three treatment regimes seemed to have a beneficial effect on the incidence of vascular complications in essential thrombocythemia. the results give a rough estimate of complication rates to be expected and may form the basis for randomized clinical trials. adhesion of activated platelets to leukocyte* has been shown to be mediated by at least two molecules on the platelet surface: p-selectin (cd62) and fibrinogen (fg) bound to the gp iib/iiia complex. interaction of platelets with teukocytes via cd62 is believed to stimulate the production of reactive oxygen species (ros) in leukocyte*. the role of platetet-bound fg as a signalling molecule in platelet-leukocyte interaction is controversially discussed: stimulatory as well as inhibitory effects on leukocyte ros production has been described. in the present study we measured ros production in undiluted samples of citrated whole blood (wb) as well as in suspensions of erythrocytes and leukocytes in platelet-rich plasma (platelet-rich wb) or platelet-poor plasma (ptatelet-poor wb). ros production was determined by measuring luminol-enhanced chemiluminescence (cl). stirring wb samples at 1,000 rpm for 10 min resulted in an activation of leukocyte* (increase of cl, upregulation of cd1 lb) and platelets (platelet aggregation, upregulation of cd62). stirring-induced cl was significantly higher in platelet-poor wb when compared to platelet-rich wb. higher cl in plateletpoor wb than in platelet-rich wb was also found when adp (moderate stimulation of platelets and leukocyte*) or fmlp (strong stimulation of leukocyte*) but not when pma (strong platelet and leukocyte stimulation) was added. dextran sulphate that is known to block cd62-mediated interaction ofplatelets with leukocyte* caused a dose-dependent inhibition of stirring-induced cl in wb samples. in contrast, when binding of fg to the platelet surface was blocked by rgds or the fg receptor antagonist gr144053, stirring-induced cl, but not cl in unstirred samples, was significantly enhanced. gr144053 also reduced adhesion of platelets to neutrophils, but did not affect cd62 exposure. the results indicate that interaction of platelets with leukocyte* in whole blood via cd62 and fg may have opposite effects on leukocyte ros production: stimulation by cd62 and inhibition by platelet-bound fibrinogen. late reocclusive processes represent one of the most commonly seen serious adverse events after coronary angioplasty. the pathophysiology of both of these processes is multifunctional and represens complex time dependent pathophysiologic events involving both the humeral and cellular processes. to test the effects of low molecular weight heparin (certoparin, sandoz ag, numberg germany) against late occlusive processes, four groups (groups a-d) of rats (7-10) were anesthetized and the external common carotid arteries were exposed. all groups received a 200 u/kg of unfraetionated heparin through the femoral vein. a 2f balloon emboleetomy catheter was inserted into the left carotid artery and passed throug the left common carotid artery leading to the aortic arck vascdar injury was induced by repeatedly (x3) inflating the catheter balloon and withdrawing it to the bifurcation. following this procedure, two hours after, equlgravimetric amounts of various agents were administered via sc route to each of the individual groups. group a was administered with certoparin od for 21 days. group b received a lower low molecular weight heprin (mr 3.4 kda). group c received unfraetionated heparin and group d served as a control. on day 21, perfusion fixation was performed on all animals. the injured and contralateral carotid arteries were excised for histelogic examinatior~ in comparison to the saline control, varying degrees of the inhibition of myointimal proliferation following arterial injury was noted in all groups. however, the degree of inhibition followed the order: heparin<certopann<llmwh_ in contrast to heparin treated group (c), the other groups a, b, and d did not exhibit any hemorrhagic lesions. however, degree of perivascular fibrosis, hemorrhage and inflammation were observed at the infusion site. in comparison to heparin, certoparin and its lower low molecular weight derivatives exhibit better efficacy in reducing balloon angioplasty induced restenosis. the observed efficacy of these agents may be due to better bioavailability and sustained duration of action. in the present paper the binding of heparin to granulocytes of human and animal species was analyzed and compared with the binding to monocytes and lymphocytes. a low-molecularmass heparin (lmmh) was covalently bound to tyramine (tyr) and subsequently tagged with fluorescein-5-isothiocyanate (fitc) by endpoint attachmenc. binding to leukocytes was quantified using flow cytometry analysis. the leukocyte populations were identified by their light scatter properties in man by cd4, cd13, and cd14 antibodies, in mice by cd 4 and gr 1 antibodies, and in rat by cd 45, ed 9, and rp 3 antibodies. granulocytes, monocytes and lymphocytes of all species bound dose-dependently lmm-heparin. the binding of lmmh-tyr-fitc ~to granulocytes was higher in human, rat, rabbit, dog, and pig as compared to monocytes and lymphocytes. mouse and sheep granulocytes did not bind more heparin than monocytes or lymphocytes. binding of lmmh-tyr-fitc was reversible in the presence of unlabeled heparin or lmmh. more than 99% of human, rat, rabbit, dog and sheep granulocyte populations could be'distinguished from monocytes and lymphocytes by their fluorescence intensity after incubation with lmmh-tyr-fitc. this separation was not obtained for mouse and pig granulocytes. the data present evidence of a specific heparin binding to granulocytes of many species and indicate the significance of fluorescent labeled lmm-heparin for biological investigations. background and purpose: ptca is an effective treatment for restoring coronary artery pat~cy; however, acute thrombotic rencclusion is a drawback of the procedure. currem prevention of rencclusion relies on antiplatelet agmts (aspirin) and antithrombin agents 0aeparin). in the present study we examined the time course of changes in blood coagulation and fibrinolytic function following ptca in patients with ischaemic heart disease. 20 patients were randomly assigned to receive either standard therapy (250 nag aspirin p.o.; 5.000 iu hepafin i.e. bolus, and 5-10.000 iu heparin intra-proeadurally)(n=10), or adjunctive therapy (aspirin, hepann; 100 mg sodium pmtosan polysulphate, spp intra-cortmarily and subcutaneously) (n=10). the sensitive markers of thrombin activation (thrombin-amithrombin iii complex ('rat), prothrombin fragmem f 1+2 (pl+2), d-dimer) and of fibrinolytic activation (plasmin-antiplasmin complex (pap), tissue type plasminogan activator (t-pa) and plasminogen activator inhibitor (pal)-i activities) were measured before, immediately after, and 6 hs after ptca. results: we found elevated mean levels of tat and fl+2 in both groups during the procedure, while plasma coneentrafiuns of d-dimer and pap remained in normal range. activity of pai-i was not elevated compared to normal comrol. the t-pa activity obtained immediately post-ptca showed significant increase in patients given spp. primary angiologic success was achieved in 80% of both groups. one patient in each group had recurrent ischaemia and emergency interventions (angioplasty, stent, bypass surgery) were performed. one patient receiving adjunctive therapy died in cardiogenic shock within 24hs arer ptca. major bleeding was observed in one patient given spp. conclusion: no significant intracoronary haemostatic activation occurred in the majority of patients during and up to 6hs after ptca. treatment with spp was associated with enhanced fibrinolytic activity. the benefits and risks of adding spp to heparm and aspirin should be evaluated by further studies, in july 19,¢1 the paul ehdich institute became responsible for quality and safety of bt~d and blood products. since ~his time, batch m~se of ppsb and factor ix prapamtions were carded out regularly, whereas ihe obligatop i testlng of all other products derived frem pooled plasma will be starling in janualy 1996. the european pharmacopoeia does .or contain directions for the quality control of ppsb preparations. therefore, ifua ~ coneenning factor ix clotting concentrates are applied, ircludmg the non activated ptt and the classical tkrombln test these m~ods are not su'rlable for reliable exclusion of ituombogenk~b/ ,of pp,sfl, because vlla actlvry is not d~. a hllhedo unsolved problem is the high sensitivity of file classical thrombin test often not correlating with ~ data of thrombogenlci~. in ordm td es~abl~ an effective quality conlrd protocol, different methods for the qual~ative detennlna~on of ila, vlla. ixa, and xa (chrornogenic, fluorege~c clolfing reactions) wera instaued. in agreement with the [deralure, considerable differences were found among the ppsb pr~ons registered in germany. cedain amounts of activated do~ng racers may be well tole~ted in redp/ents. ~no are in a steady state condition. however, in severely ill patienls, eg. post operatively, tissue factor may be expressed which pofenllatesthe dsk of ~aclor vlta. in the case of a certain ppsb preparation hlgh ¢onceatratio~ of acthrated cto~ng factors, especially ~vated factor vii, cotmlaled with fatal comlffcal~ns, evaluating ~ expe~ concenlralbn limits for adjvated prothrombin co~np~.x factors as prerequisite for ~e batch r~ease have to be applied. aprotinin is used as a hemostatic agent in cardiopulmonary bypass surgery and other indications where hemostatic compromise results in hemorrhagic manifestations. the mechanism by which this agent produces hemostatic effects is not known, however, inhibition of serine proteasas such as plasmin, kallikrein and dastase and modulataion of platelet receptors have been considered as possible target sites. to determine the effect of aprotinin on tissue factor mediated activation of human platelets, whole blood samples were drawn at baseline and 24 hours aider aspirin (650 mg po) ingestion (n=5). blood samples were supplemented with saline and aprotinin (40 ug/ml) and tissue factor activation was studied at 200 pg/ral. platelet activation was investigated using gtycoprotein ffla and gmp 140 specific antibodies on a flow cytometer (epic coulter). tissue factor was found to produce the activation of both the baseline and asp' "mnized platelets. aprotinirt augmented the tissue factor activation of platelets in both the control and aspirin supplemented groups. however, the relative increase in the aspifinized group was much monger (25% of the baseline). these results suggest that aprotinin augments the tissue factor mediated activation of platelets in both the normal and aspirinized individuals. while these effects may result in hemostatic restoration in aspirin compromised patients, in normal individuals the clinical implications of this effect remain to be clarified. , 10-20 (group 2) or >20 years (group 3) duration. anticoagulated whole blood was incubated with fluorescent antibodies to gpib and gmp-140 (two colour method) and analyzed with a flow cytometer. thrombomodulin, f1+2, protein s, 13-thromboglobulin were measured according to standard procedures. results: surface expression of gmp-140 was not different in groups 1 to 3, however, there was a tendency to higher acitvation in group 1 (<10 years iddm). results for thrombomodulin, f1+2, protein s, 13-thromboglobulin will also be presented. conclusion: though it did not reach statistical significance, platelet acitvation seems to be more important during early diabetes. this wilt be correlated with endothelial and plasmatic activation markers. in our clinic four patients with hiv-related thrombocytopenia were treated with a lot of gammagard (93f21abllf), which later turned out to be hcv contaminated. before infusion all patients were negative for hcv antibodies and hcv rna. 2 to 8 months after infusion 2/4 patients, who suffered from arc at the time of hcv infection with cd4 counts >100/pl, seroconverted, whereas in the two other patients, who suffered from aids with cd4 counts below 100/pl, there was no seroconversion. in all cases hcv rna was found. genotyping with inno-lipa (innogenetice) showed hcv genotype l(b) in all patients. liver enzymes and hcv rna copies were measured repeatedly over a period of one year after infection. the 2 patients with arc showed a strong increase of hcv rna titre during the first 3 to 4 months after infection, followed by a rapid decrease within the next months. in the patients with aids hcv rna copies increased moderately within the first 4 to 6 months, followed by a slow decrease. elevation of liver enzymes was mild in the aids patients and seems to be independent from the hcv rna titre. in the arc patients liver enzymes changed parallel to hcv rna titers with a delay of 2 to 3 months. the course of hiv infection was only slightly influenced by the acute hepatitis c as measured by cd4 counts, i%2microglobulin and hiv rna copies. introduction:mechanisms underlying ischemia/reperfusion injury have been thoumughly investigated in experimental models. leucocytes appear to play a main role through production of cytokines and overexpresssion of adhesion molecules. in experimental animals, administration of monocional antibodies (mab) recognizing cd18 can reduce organ injury following ischemia/repedusion. no data, however, have been reported concerning clinical ischemia situations. patients and methods:we investigated expression of cdt8, cd1 la, cdf l b and cd1 lc in granulocytes, monocytes and lymphocytes from peripheral blood of five patients undergoing elective hand surgery. the tourniquet was applied on the upper arm and heparinized samples from cubital veins were obtained before and at the end of ischemia. control samples were drawn from the nonischemic contralataral arm with the same timing, duration ot ischemia ranged between sixty and one hundred minutes (80~16). whole blood samples were incubated with specific, fluorochmme labelled antibodies and analyzed by fluorocytometry (facscan, becton dickinson, san jose, ca). mean fluorescence intensity (mfi), quantitatively reflecting surface expression of the indicated markers was evaluated for the individual cell populations. data were compared by the paired student's t-test, p<0,05 was evaluated as significant. results:mfi for all markers was comparable in all cell populations in samples obtained before ischemia from both arms. in contrast, expression of cd18 was significantly enhanced in granulocytes (321_+50 vs. 189_+38), monocytes (653-+54 vs. 426+122) and lymphocytes (299_+45 vs. 228-+36) from samples derived from the ischamic arm, as compared with the nonischemic arm, as measured at end of ischemia. at the same time, an increase of cdf lb on granulocytes (500~_342 vs. 213+150) and monocytes (533+359 vs.237-+206) but not on lymphocytes was found, no modifications of cdlta and cdttc expression could be observed. there was no correlation between duration of ischemia and quantitative expression of these markers, conclusions:our data indicate that relatively short ischemia periods induce an increased expression of ~2" integrins adhesion molecules on leucocytes. these results suggest, at close similarity with findings from expodmental models, that overexpression of adhesion molecules might play an important role in the induction of ischemia/reperfusion injury, in humans. in patients suffering from chronic inflammatory bowel diseases, such as morbus crohn and colitis ulcerosa, we observe massive, sometimes barely staunchable bleedings. hereby, the deficiency of coagulation factors, especially of factor xiii in plasma is established. ttowever the influence of factor xiii on the pathomechanism of the underlying disease is still under discussion. therefore we studied the f xiii content in the intestinal mucosa. an immunohistochemicat method was developed using commercially available antibodies against f xiii subunit-a, the detection of mucosal factor xiii depends on the amount of chromogen bound to the antibody-horseradish-peroxidase complex. with this method, it is possible to locate but not to quantify f xili in the intestinal tissue. therefore we developed an elisa-metbod in homogenized intestinal tissue, using commercially available antibodies. its precision was validated using a standard curve with commercially available factor xiii preparations (fibrogemmin®). the detection limit of this method is > 0.05 i.u. f xiii/ml of tissue solution. freezed dried intestinal tissue (lmg) was homogenized in 1 ml buffer using a potter. specimens of the large bowel revealed f xiii values of 0,21 + 0,0038 i.u. (x __+ sd), tissue solution. with this method it is possible to quantify tissue-bound faxtor xiii. studies are in progress to elucidate the content of f xiii in the intestine of patient's suffering from infammatory bowel diseases in order to contribute data to the pathomechanisms of f xiii deficiency. in a previous double-blind, controlled trial we were able to show that aprotinin administration has significantly contributed to reduce periand postoperative bleeding complications without increasing the risk of thromboembotic complications. the question arises whether this beneficial effect may be associated with its effects on intraoperative fibrinolysis. therefore, 20 patients were treated with or without aprotinin (2 million kiu loading dose over 15 minutes followed by 500,000 kiu per hour), and citrated blood samples were obtained at the following time points: before operation, after induction of the anesthesia, at the beginning of operation, intraoperatively when the femur shaft was implanted, and 24 hours postoperatively. the determinations of plasmin/antiplasmin-complexes, d-dimers, thrombin/antithrombin iii-complexes, and prothrombinfragments 1 +2 were performed by means of test kits from behring, germany (enzygnostrpap micro, enzygnost r d-dimer testkit, enzygnost r tat micro and enzygnost r f 1 +2 respectively). -all markers of activated fibrinolysis and blood coagulation were significantly increased in the groups with and without aprotinin treatment, the highest activities to be seen when the femur shaft was implanted. however, the values of pap and d-directs of the aprotinin group were below the values of the control group until the end of operation. the markers of activated coagulation showed the opposite effect, however the differences between the two groups were not significant. as expected, the aptt was significantly prolonged in the aprotiningroup. the aprotinin treatment was also associated with a significantly lower blood loss in these patients. -concluding it can be said it is not clear whether the blood saving effect of aprotinin may be exclusively attributed to its antiplasmin activity since the differences of the fibrinolysis parameters were not statistically significant. further blood samples should be analysed between the implantation of the femur shaft and the end of operation. in our laboratory large amounts of human prothrombin are required (30-50 mg/week). as we try to produce meizothrombin and meizothrombin-des-fragment-1 from human prothrombin and to apply it as an antidote for hirudin, the classical adsorption to barium sulphate or aluminum hydroxide from human plasma cannot be used. commercially available human prothrombin is expensive and of an unacceptable quality for our applications. in most of these batches we found small amounts of factor x and prothrombin activation products. we now developed a procedure to isolate prothrombin from "prothrombin complex concentrates" (ppsb-250-bulk, drk-blutspendedienst nds.). the concentrate also contains fac-tor vii, factor ix, and factor x. the prothrombin had to be separated from these factors. the concentrate we used contained amounts of other proteins and activation products of prothrombin (e.g. prethrombin-1) as well. for the preparation of prothrombin from ppsb we used anion exchangechromatography (resource-q ®) on an fplc ®. we applied dissolved ppsb directly or after buffer exchange on sephadex g-25 onto the column at room temperature. the prothrombin was eluted with an naci-gradient in trisodium citrate buffer, ph 7.0. the buffer conditions are similar to the conditions used in the preparation of ppsb. the quality of the prothrombin so obtained was sufficient for most of our experiments. a second purification step on ion-exchange resulted in a 99% pure product devoid of contaminating factor activities and activation intermediates as examined with coomassie and silver stained sds-page electrophoresis and assays for factor x. this prothrombin contained full enzymatic activity and its activation by specific snake venom prothrombin activators showed the known activation products. we are now able to isolate the amounts of pure prothrombin required for preclinical investigations. most of the commercially available lmwhs such as enoxaparin, fraxiparin, and fragrnin are prepared by chemical methods which can result in desulfation and other chemical modifications of the internal structure leading to differences in the pharmacologic effects. on the other hand, tiactionated lmwhs retain their native characteristics and are structurally similar to heparin. in addition, the oligosaocharide sequence responsible for atiii binding is not modified. physical methods such as gamma irradiation (~co) have been used to fi'agment sulfated glycosaminoglyeans yielding fragraents without chemical modifications (deambrosi et at. in : biomedical and biotechnological advances in industrial polysaccharides, pp. 45-53). utilizing this technique, depolymerized heparius exhibiting different molecular weights can be obtained. this communication reports on the biochemical and pharmacologic effects of several such depolymerized heparins to demonstrate the molecular weight dependence on biologic activity. fragments exhibiting molecular weights of 5, 7, 8, and 9 kda were prepared by exposing concentrated heparin solutions to a rectilinear gamma ray beam at intermittent doses of 2.5 to 25 mrad under controlled temperatures. unlike the chemically depolymerized heparins, these fractions did not exhibit any decrease in charge density or atiii affinity. in routine assays for heparin, a clear cut molecular weight dependance on the anticoagulant and antiprotease actions was observed. on a gravimetric basis, these agents produce superior antithrombotic actions in comparison to chemically depolymerized derivatives. these studies suggest that gamma irradiation can be used to prepare lmwhs which retain their molecular integrity and therefore may prove to exhibit a more comparable biologic profile to hepari~ futthermore, lmwhs produced by gamma irradiation lack the usual double bond fommtion which requires the use of additives which can alter the product profile. university hospital, dept. of angiology, frankfurt a.m., germany introduction: thromboembolic disease constitutes a major clinical problem and among others a defective fibrinolytic system has been suggested as a predisposing factor for the development of thrombosis. the plasma fibrinolytic system can be impaired by inherited deficiencies of plasminogen defective release from the wessel wall tissue plasminogen activator (t-p'a) or by high ptusma levels of regulatory proteins, such as plasmino-8en. activator inhibilors (pal). the aim ....... of the present study w~s to eshmate the prevalence of decreased fibnnolyl~c actwlty m young pls. with thrombophilia. patients: a great population of 884 pts. (fenmle 478, male 406; age 21-61 ys median 39.8 ys) with venous thromtx~emolism before the age of 45 years were investigated in regard to their plasma fibrinolytie system. in none of them well established thrombophilia risk factors could be identified previously. methods: plasminogen ~behdngwerke), pai-1 activity (ehromogenic assay, biopool), pal-i anugen coneentration (elisa, biopool), t-pa activity (chromogenic assay, biopool) and antigen concentration (elisa, biopool) were measured before and after venous oeclusion.vo was performed z 12 month after the last thromboembolic epi~xle. 24 healthy subjects (median age 24.7 ys) served as controls. results." 24 pts.(2.7%) were classified as plasminogen deficiencies (activity and antigen). 142 pts.(16%) had significantly elevated levels of pal activity (up to 120 u/ml) and pal antigen (up to 90 ng/ml). none of the pts. with high pal levels had laboratory signs of acute phase reaction. low t-pa activity could be demonstrated and confirmed in 121 pts., aecordingto a prevalence of 13.6% (range: 0-2.7 u/ml; reference limils: 2.8 -21.8 u/ml). however, there was a significant negative correlation between t-pa activity and pal values. in 67 pts. (55.4%) the low t-pa activity was associated with increased pal levels whereas the t-pa antigen concentration was normal. a parallel reduction of t-pa activity and t-pa antigen (range: 0.35-3.5 ng/ml; reference limits: 3.6 -21.0 ng/ml) were determined repeatedly in 54 pts. (f 23, m 31, median age 39 ys). thus, the prevalence of a defective t-pa release was 6.1% in our study group. conclusion." in comparison to the frequency of inherited deficiencies of other well established thrombophila risk factors we have observed a relativel~ high prevalence of diminished t-pa activity, elevation of pal respectively in our study group. our data strongly indicate that besides t-pa and pal acuvity, antigen concentration for both parameters should be determined in pts. with thrombophilia. the antithrombotic and anticoagulant effect of the supersulfated low molecular weight heparin ssh 14 was studied after i.v. and s.c. administration in rats. thrombus formation in the jugular vein was induced by i.v. injection of activated human serum and following stasis for 20 rain and was assessed by a thrombus score ranging from 0 (no thrombus formation) until 3 (complete thrombus formation). ssh t4 injected either 10 min (i.v.) or 30 rain (s.c.) before thrombus induction caused a dose-dependent antithrombotic effect in a range from 0.25 to 2 mg/kg i.v. and 1 to 4 mg/kg s.c. there were clear differences in the antithromboric effectiveness between female and male animals, i.e, in female rats antithrombotically effective doses were lower than in male rats (edh0 after i.v. injection in females 0.35 mg/kg, in males 0.9 mg/kg). the sex differences were confirmed in studies on the time course of the antithrombotic effect. after i.v. injection of fully effective doses (2 mg/kg i.v. and 4 mg/kg s.c., resp.) the antithrombotic effect disappeared after 8 h in female or after 4 h in male rats. for studies on the anticoagulant action blood was drawn from the femoral artery and after centrifugation global clotting assays were performed in plasma. similar to its antithrombotic action ssh 14 also caused doseand sex-dependent anticoagulant effects. the most sensitive assays were the aptt and the heptest; thrombin time and prothrombin time were less or not influenced by ssh 14. in conclusion, ssh 14 was found to be an effective anticoagulant and antithrombotic agent in experimental studies in rats. at present there is no explanation for the clear sex differences found in this species. venous thromboembolic disease is the most frequent complication in patients undergoing total knee replacement therapy. patients and methods: after informed consent 3x30 patients were included in an open randomized clinical study and the incidence of venous thromboembolisrn was examined using different regimes for heparin prophylaxis (30 patients received fraxiparin 36 rag once daily, 30 patients clexane 40 once daily and 30 patients 7500 u calciparin twice daily). there were no differences between the groups concerning age, sex, body weight, risk factors, surgeons, decrease in hemoglobin~ and requirements for blood products. pre surgery, day 1, day 5-7 phiebograms were performed and also tat, dimers, fl+2 prothrombin fragments were examined. results: 1., dvt in 26 patients (28.9%). dvt in 5/30 patients under calciparm prophylaxis, 8/30 patients under fraxiparin and 13/30 patients under clexane treatment. 2., low speciflty (3.4%) of dimers and tat (24%) for detecting a dvt in these special patients undergoing knee replacement therapy, elevated fi+2 fragments in the dvt group at ti and t2 vs the patients without dvt (t1 dvt: 3.24+-1.8 vs. 1.6+-0.3 -p= 0.0042). 3, only 8/26 patients (31%) with dvt had clinical signs of thrombosis. conclusions: 1., there is an increase of thrombin gneeration measured by tat and dimers after knee replacement therapy. there are further studies with more patients necessary to confirm that fl+2 prothrombin fragments can discriminate between patients with and without dvt from a clinician's point of view. 2., phlebographicauy confimled dvt in almost 30% of our patients demonstrate the high thromboembolic risk in these patients. von willebrand's disease (vwd) type 2 is characterized by absence of high molecular weight muitimers. qualitative changes in the structure of the molecule might be associated with enhanced binding of von willebrand factor (vwf) to platelet glycoprotein lb. therefore in some patients vwd type 2 is associated with severe thrombocytopenia. here, we report on a 9 year old boy who presented with severe purpura and platelet counts about 20000/gl at the age of 2 years. thrombocytopenia did not respond to corticosteroids. a normalized platelet count of short duration was observed after high-dose immunoglobulins. in addition, increase of platelets was seen after anti-d treatment. thus, although platelet associated antibodies were not detected, thrombocytopenia seemed to be caused by an autoimmune mechanism. despite platelet counts above 50000/gl, the patient experienced severe bleedings with a significant decrease of hemoglobin levels. therefore, he needed several transfusions. coagulation analysis revealed vwd. application of ddavp lead to a normalization of partial thromboplastin time (ptt) and an increase of factor viii with subsequent cessation of bleeding symptoms. recently, vwd was typed 2 by lack of high molecular weight multimers. in conclusion, we report a case with vwd type 2 responding to ddavp. however it is unclear, whether thrombocytopenia is part of the vwd type 2 or of autoimmune origin. since autoimmune antibodies have not been detected, the effect of immunoglobulin treatment might be explained by blockade of enhanced binding of vwf to glycoprotein lb. von willebrad disease (vwd) with a prevalence of 0,8% (ruggeri 1994, rodeignere 1987) seems to be the most frequent inherited hemostatic disorder. • the diagnostic criteria for vwd are clinical picture, family hostory, laboratory findings: bleeding time, partial tromboplastine time (ptt), level of factor viii:e, vwf, vwf:ag, ristocetin induced platelets aggregation (ripa) and multim~-analysis.the diagnosis ofvwd is occasionally difficult, especially in early childhood because the laboratory data may vary due to time of investigation, as well as abnormalities may not be present in all sub-types the aim of this study was the evaluation of diagnostic approach to vwd in childhood and diagnostic reliability of all available laboratory tests. all previously mentioned laboratory tests have been done on our own material (51 child who satisfied all criteria for vwd, 23 boys and 28 girls, 1-9 years old) except mulfimer analysis which was unavailable in some cases. majority of laboratory tests proved to be highly specific and necessary for diagnosis. however, the diagnostic reliability of fviii:c and adhesion of platelets is much lower in mild cases in comparison to total sample, while ptt is an unvaiied test. the most specific screening test for vwd is vwf which diagnostic reliability is almost 1,00. the optimal strategy to establish general diagnosis of mild forms ofvwd is use of vwf and vwf:ag plus ripa if necessary and multimer analysis to classify variant types. we report on a new multimeric structural defect of vwf detected in a german family (two sisters and their three children): all members of the family who presented to our outpatient clinic had an increased spontanous bleeding tendency (moderate or strong hematoma, epistaxis, menorrhagia). prolonged bleeding could be observed after surgical procedures (adenotomia, tooth extraction) and after trauma (laceration). wound heeling was impaired in two cases. clotting assays showed slightly prolonged apti" and a mild decrease of f viii:c, vwf:ag and vwf:rcof levels. collagen binding activity was within normal ranges. bleeding time (simplate i) was slightly prolonged. the analysis of the multimeric structure in plasma showed quantitative and qualitative abnormalities: all multimers were detectable; the structure of vwf was reproducably abnormal in all family members so that the defect must be caused genetically. the thmmbocytic vwf showed neither qualitative nor quantitative alterations. minirin@ (ddavp) was administered as a test dose of 0,3 ~tg/kg bw in 100 ml 0,9% nacl-solution i.v. to evaluate efficacy and tolerance: clotting assays showed normalization of a_vrt, f viii:c, vwf:ag, vwf:rcof in plasma and shortening of bleeding time in three cases. an insufficient rise of vwf:ag and vwf:rcof levels could be observed in one case. one patient had no rise of f vm:c but a corrected bleeding time. multimeric analysis showed no structural change. the administration of ddavp was well tolcrated in all cases. the existance of all multimers in plasma and the normal collagen binding activity suggest that the structural abnormalities of vwf in this family does not cause functional defects so that the defect could be classified as a type i vwd. the response to ddavp was only partially effective. mild von willebrand disease (vwd) is far the most frequent congenital bleeding tendency. its diagnosis is very helpful in pre-operative check-up in order to avoid bleeding complications during surgery. following post-operative periods or monitoring the management of haemorrhagic episodes in vwd patients is also strongly recommended. current methods involve complex technologies, are time consuming and require large series. these assays lack the expected flexibility for rapid individual testing in patients. a new and flexible assay which works on the fully automatic walk-away coagulation instrument, sta, has been developed for these applications (liatest vwf). the technology is an immuno-turbidimetric method using mierolatex particles coated with rabbit polyelonal antibodies specific for vwf. the assay has a dynamic range from 2 to 420% yon willebrand factor (vwf) concentration, it works with a 2 fold dilution of tested plasma (50 td) and it offers a calibration established with the nibsc international standard. the total assay time is of less than 10 minutes and the detection threshold is of 2% there is no prozone effect up to concentrations higher than 1,000% vwf. intra-assay reproducibility is < 4% and inter-assay one < 5%. in dilution studies a mean recovery of 98% was obtained. in a study on 55 plasma samples from norma~ individuals, patients with high vwf concentrations, and vwd, comparison with the elisa technique demonstrated a correlation coefficient of 0.997 with a slope of 0.978 and an intercept of 3.30. in the low assay range too, a good agreement was obtained with the elisa. we conclude that liatest vwf is a reliable, flexible, sensitive, and rapid automated assay which fits well the vw'f assay applications in coagulation laboratories. fibrinolysis, the process during which the active enzyme plasmin is generated in a regulated and localised way, is -in a classical understanding -responsible for the dissolution of blood clots formed in a vessel. for this activity, t-pa is generally assumed to be the most important plasminogen activator and its activity, is regulated by enzyme kinetic mechanisms dependent on the presence of fibrin. with this background t-pa is used for thrembolytic therapy with great success. however, data from t-pa knockout mice indicate that t-pa might not be responsible for inhibiting the spontaneous development of intravsacular thrombi but only for dissolution of fibrin formed upon a coagulation challenge. in contrast, u-pa, generally assumed to be important for extravascular proteolytie activity on activated or tumour cells, seems to lead to the development of spontaneous fibrin formation in a mouse knockout model. on the other hand, the major plasminogea activator inhibitor pal-i seems not only to regulate intravascular fibrinolysis but seems to also be important for the progression of vascular diseases (neointima formation is e.g. increased in a pai-1 knockout model, but increased levels of pai-1 seem to predict reocclusion after angioplasty). in addition to their functioning as enzymes and inhibitors, components of the fibrinolytic system seem also to be involved in signalling processes in tumour and other cells. the u-pa/u-pa-receptor system could be shown to function as a chemotactic system and to elicit a migratory and mitogenle response in monoeytes and tumour ceils as well as in vascular cells. for such a response activation of tyrosine kinases of the sre-family might be responsible in some cell lines, but other signal transduction pathways e.g. involving caveolae and the starprotein can not be excluded. there seems to be a further important role of components of the fibrinolytic system which involves serine protease inhibitors (serpins): serpins have homologies to hormone binding proteins and cleavage of serpins by their target enzymes not only leads to inactivation of the enzyme but also to a possible release of bound hormones from the serpins. from these data clearly the relevance of any regulation of the fibrinolytie, system depends on the specific function of the system to be dealt with. in addition to "fibrin binding", "receptor mediated" and "genetic control" (e.g. 4g vs. 5g in the pai-i promotor) also "signal transduction" and "hormone delivery" are distinct functions of the system with specific regulation. plasmatic for both, healthy persons as well as for patients with angina pectoris it could be shown that increased values of plasma fibrinogen, factor viic and vwf:ag are significantly associated with the risk to suffer an acute myocardial infarction or cardiac sudden death. the same holds for tpa:ag. however, a group analysis in quintiles reveals that particularly low tpa:ag values are connected with a particularly low coronary risk. unexpectedly also the acute phase protein crp is positively associated with increased coronary risk. for clinical purposes these factors have already been included into coronary risk scores in order to improve the individual risk prediction in combination with lipids and other risk factors. the assessment of the pathophysiological significance of these observations remains at dispute. 4 pathways are discussed: 1. the assumption that increased plasma values of those factors indicate increased coagulation activity could so far not be established in prospective studies. 2. both vwf:ag and tpa:ag are produced in endothelial cells. an increase of their plasma level could therefore indicate increased endothelial cell functions which accompanies progressive atheromatosis. the risk association of the two acute phase proteins crp and fibrinogen could be interpreted analogously. 3. first prospective studies favour the assumption of a genetic determination to an increased production of coagulation proteins in persons at particular coronary risk. it could also be shown that there is a certain dependance of the gene-polymorphism for co-and 13-fibrinogen chains from the coronary risk. 4. even slightly elevated concentrations of fibrinogen and/or vwf:ag may influence the quality of a coronary thrombus both by increased physical stability and by reduced fibrinolytic lysibility. this could mean that an early coronary clot under these conditions could more readily develop to a stable, occlusive thrombus. a newborn with pronounced bleeding tendency had a prothrombin (prth) deficiency below 2.8% in a clotting assay. both parents had activities of 71% and 69%, respectively. however, the immunological determination ofprth by elisa revealed normal concentrations in all family members (93%-101%). furthermore, thrombin generation as investigated by a chromogenic assay using ecarin for activation of prth was normal as well. activation of prth by fxa was investigated by reealcificafion of the plasma samples and further analyzed for prth and its derivatives produced. although clotting times still were different, finally, normal levels of fl+2 and tat were generated as determined by elisa. western blot analysis using polyclonal (rabbit) antibodies to prth and a monclonal antibody specific to human thrombin, revealed different patterns of prth degradation products. tat was only weakly visible in the serum of the mother and nearly absent in the child.the mobility of prothrombin and thrombin was different compared to normals indicating a lower molecular weight. after reduction of disulfide bridges a higher molecular weight of thrombin was observed compared to normals indicating an insufficient cleavage ofprth and formation ofprethrombin 2. these observations let suggest that prothrombin marburg is a deletion mutant lacking the cleavage region arg 320-ile321. upon cleavage by factor xa only prethrombin 2 is formed under liberation of fl+2. this prethrombin 2 is able to cleave chromogenic substrates in the ecarin assay. probably, prethrombin 2 forms a complex with atiii which is detected by elisa, but unstable under denaturing conditions as in the western blot. as a major complication of haemophilia a treatment, up to 30% of the severely affected patients develop antibodies to substituted factor viii. investigating 133 patients and considering the data of further 231 patients of the haemophilia database, we could show, that risk of inhibitor developement depends on the patient's mutation type. patients with more severe gene defects, like intron 22 inversions, stop mutations or large deletions had a risk of about 35% for inhibitor developement, which was about 7 times higher than for missense mutations or small deletions. besides an influence of mutation type, we investigated other parameters e. g. immune response genes (i-ila-genotype) and clinical aspects (treatment onset and frequency, type of concentrate) that might also affect inhibitor formation. to exclude any effect of mutation type, we focussed on 72 patients with an intron 22 inversion. hla-typing showed that some t-ila-alleles (dqb0602, bt) occurred more otten and others (dqa103, dqb603, dr13, c2) less frequent in inhibitor patients. treatment onset, frequency and type of concentrate apparently do not affect inhibitor incidence. the results presented here, prove that inhibitor development is considerably influenced by the mutation type. this supports the hypothesis that patients with severe molecular defects have no endogenous factor viii protein and that substituted factor viii represents a foreign protein, leading to an immune response, e. g. the production of alloantibodies. in addition, the immune response seems to be modified by the hla-genotype. however oar findings (in terms of genotype and treatment parameters) can only explain part of the inhibitor pathogenesis. it is still unsolved why substituted factor viii does not lead to a recognizable immune response in 2/3 of the patients with severe molecular factor viii gene defects. consequently other factors, probably concerning the antenatal phase, must be involved. viia in the treatment of patients with inhibitors against factor viii or ix: a german/swiss/austrian multi~center trial d. ellbriiek*, i. scharrer**, j. dethling***, and the rfviia study group *section h~mostaseology, university ulm **dept. of angiology, jwg-university hospital frankfurt a.m. ***novo nordisk, mainz administration of activated recombinant factor vii (rfviia) can by-pass the fvnlwlx pathway and offers an alternative treatment for patients with antibodies (inhibitors) against these factors. from november 1994 to october 1995, a total of 25 bleeding episodes and 10 surgical interventions in 18 patients were treated with rfviia in a phase iiib multicenter trial. diagnosis was hemophilia a (n = 15) or b (n=l) with inhibitor, and acquired inhibitor against factor viii (n=2). various serious bleeds, from complicated joint and gingival bleeds to lifethreatening psoas bleeds, have been treated. operations have been tooth extractions, radiosynovectomy, implantation and explantadon of porth-acaths and one adenotomy. dose regimen was 90-120/zg/kg bw every two to three hours until clinical improvement, with subsequent dose reduction. results: for bleeding episodes, response to rfviia after 24 hours was effective in 72%, partially effective in 12 9"0, ineffective in 129"o and not evaluable in 1 (4%) of the patients. two of the three treatment failures were associated with very long dosage intervals of rfviia. the third patient was in a critical situation with artificial high pressure respiration and polytransfusion because of a hematothorax, and suffered a terminal intracerebral bleed. the efficacy of rfviia for surgery was very good. response to treatment was independent of antibody titer. no signs of dic or activation of coagulation were noted. conduslon: in our experience, rfviia is an efficient and safe treatment for inhibitor patients with acute bleeding episodes. it should be investigated, whether rfviia can be an alternative treatment also for the hometreatment situation. successful immunetolerance therapy of f vih-inhibitor in children after changing from high to intermediate purity f vih concentrate w. kreuz, j. joseph-$teiner, d. mentzer, g. auerswald*, t. beeg, s. becker zentrum der kinderheilkunde, j. w. goethe-universit~itj frankfurt am main *professor hess kinderklinik bremen introduction: inhibitor to f viii is the most severe complication in treatment of patients with haemophilia a. the incidence of f viii inhibitors is estimated to range between 15-33%. several authors reported that the immunetolerance therapy (itr) of f viii-inhibitors can be induced with high dose f viii concentrate. objective: this presentation will show data of four children with haemophilia a and f viii inhibitor (high responder), who had an unsuccessful lit with high dose f viii concentrate (high purity) in the first step. f viii concentrate was changed to an intermediate purity product (haemate hs®) in the subsequent course of h't. all patients received bleeding prophylaxis with an activated-prothrombin-complex-concentrate (feiba®). results: median age was 13 (9-18) months, when the inhibitor was first detected. in all four patients the f viii inhibitor titre increased under immunetolerance treatment with f viii concentrate (high purity) in the first step of therapy. after changing the f viii concentrate (intermediate purity) the inhibitor titres decreased continuously after a rebooster effect to 0be within months. median duration of f viii inhibitor elimination time (until first testing of 0 be) was 3 (2-5) months. in all patients the f viii inhibitor was successfully eliminated. until now all patients are under prophylactic treatment with f viii concentrate and had no positive inhibitor testing since. median observation time since the first testing of 0 be is 14 (4-60) months. conclusion: different studies concerning immunetolerance treatment have been successful with f viii concentrates of different purity. according to our experience in these four presented patients, we assume that probably not the purity of the f viii concentrate is important for the induction of immunetoleranee, rather than the type of f viii presentation in the used concentrate. the used preparation (haemate hs®) is a f viii concentrate with high concentration of vwf, which is known to be important for the protection of f viii against degradation by proteases. this may be a mechanism for a prolonged antigen presentation to the immunesystem and thus may have a positive impact on the outcome ot itr. long scale trials are needed to prove the above assumptions. thrombasthenia glanzmann is a disease affecting platelet function because of a partial or total lack of glycoprotein (gp) ilbllla expression or a modification of this complex. since the receptor dysfunction goes along with reduced or absent platelet aggregation and adhesion, it causes bleeding complications in case of injury. here we report about a 60 years old women, who suffered since early childhood from a severe bleeding disorder. life threating bleeding complications occured after tooth extraction and after abdominal surgery. analysis of the patients platelets revealed normal values for the platelet count, whereas their volume showed to be increased (11 fl). clot retraction was diminished to 17%. platelet adhesion to siliconised glass and human subendothelial matrix was reduced, as was the spreading of the platelets. adp (i#m) induced platelet aggregation was inhibited, while collagen-, ristocetin-and thrombin-induced aggregation showed to be normal. cross immunelectrophoresis resulted in an atypical peak of gpiibllla with reduced electrophoretic mobility. in the electroimmunoassay according to laurell 14% of gpiibllla was detected. moreover we observed a markedly diminished 125j-fibrinogen binding. sequence analysis of the gpiib and gpiila cdna after pcr amplification unraveled a g2508 --, a transition in gpiib, substituting gly805 --* glu. the structure/function relationship of this mutation has still to be investigated. we report two new abnormal fibrinogen variants, denoted as bem iv and milano xi, both having an exchange of arginine to histidine in position 16 of the ac~-chain. routine coagulation studies revealed prolonged thrombin and reptilase clotting times, low plasma fibrinogen concentrations determined by a functional assay but normal fibrinogen levels measured by the immunological assay. the onset of turbidity increase following addition ofthrombin to purified fibrinogen was markedly delayed in both variants. release of fibrinopeptide b by thrombin, measured by reversed phase hplc, was normal whereas only one half amount of normal fibrinopeptide a was released. in addition to normal fibrinopeptide a, an abnormal fibrinopeptide a* was cleaved from both dysfunctional fibrinogens. the structural defect was determined by asymmetric pcr and direct sequencing of a gene fragment coding for the nh2-terminus of the aachain. both variants were found to be heterozygous for the transition g to a at nucleotide position 1203, leading to the substitution actl6 arg-->his, resulting in a delayed fibrin polymerization. the simple assay permits detection of the most common amino acid substitutions occuring in the nh2-terminus of the ac~-chain of the functionally abnormal fibrinogen variants. protein c inhibitor (pci) a member of the serpin family is also known as plasminogen activator-3 (pal-3). pci was first described as a component of human plasma, regulating the activity of activated protein c and other sedne proteases of the human coagulation and fibdnolysis system. since then pci was found to be present in extra-plasmatic systems also. high concentrations of pci were detected in human seminal plasma suggesting a role for pci in human fertility. significant concentrations of pci mrna and antigen were located in lysosomes of proximal tubular kidney cells suggesting an intracellular function for pci in this environment. in this study we present evidence that pci is also present in human pancreas. rna from human pancreas was reverse transcribed and pcr amplified. the resulting pci cdna was identical with pci cdna from human liver. ~p labeled antisense rna probes used in in situ hybridization experiments with human pancreas tissue sections showed that pci rna was located in the acinar ceils. pancreatic fluid was analyzed by sds-page and immunoblotting. using monospecific antibodies directed against human plasma pci, a 57 000 mw protein band was observed which comigrated with purified human plasma pci. our results show that pancreas cells contain a significant concentration of pci mrna. this message is localized in the secretory acinar cells. therefore we conclude that pci antigen found in pancreatic fluid is likely to originate in the pancreas. the role of pancreatic pci is unknown at present. however, since thrombosis and systemic hypercoagulable states are known complications of pancreatic diseases our results and in vitro experiments by others showing that pci can inhibit pancreatic enzymes such as chymotrypsin and trypsin indicate that pci may be part of the inhibitor potential which protects pancreatic tissue from auto degradation. these inhibitors normally prevent the release of active pancreatic proteases into the vasculature or microcirculation where destabilization of the coagulation balance and subsequent thrombus formation could occur. institute for clinical chemistry and laboratory diagnostics and *clinic for cardiology, universi w of duesseldorf p-selectin (cd 62p, the former granule membrane protein 140 or gmp 140) is an integrated membrane protein of platelets and endothelial cells. under inactivated conditions it is stored in the alpha granules of platelets and in the weibei-palade bodies of endothelial cells. endothelial cells covering atherosclerotic plaques show an increased expression of p-selectin. 13-thromboglobulin (13-tg), which is also expressed from the alpha granules of platelets during adhesion or aggregation, is regarded as a marker of platelet activation in vivo. coronary thrombosis plays a central role in the pathogenesis of acute coronary syndromes. we therefore analysed cd 62p and 13-tg in acute coronary syndromes, healthy subjects (hs, n=l i), patients with stable angina pectoris (sap, n=20), unstable angina pectoris (uap, n=l 2) and acute myocardial infarction (ami, n= 12). plasma samples were obtained by using ctad vacutainer tubes (0.109 m na~-citrate, theophylline, adenosine dipyridamole). patients with cad showed significantly increased plasma concentrations of cd 62p (hs: 98+20 versus sap: 133+38ng/ml, p<0.05; versus uap: 128+28 ng/ml, p<0.01; versus ami: 144+72 ng/ml, p<0.05) independent of the severity of clinical symptoms. in comparison only patients with ami showed significant higher 8-tg concentrations compared with hs (hs: 30+20 versus ami: 39+14ng/ml, p<0.05). although the cd 62p plasma concentrations showed no relationship to the clinical severity, hence there was a positive correlation between cd 62p (r=0.47; p< 0.001; n=55) to the severity of cad classified as i, 2, 3 vessel disease. it is concluded that elevated cd 62p concentrations are correlated with the severity of cardiovascular disease. cd 62p is not suitable for differential diagnosis of acute coronary syndromes, because it is elevated independently of the clinical status of the patients. the involvement of platelets in the pathogenesis of acute myocardial infarction may be indicated by the increased 13-tg concentrations. iklinik nr herz-, thorax-und herznahe gef&schirurgie und 2institut x~tr klinische chemie und laberatodumsmedizin der universint regensburg an increased blood loss following surgery with extracorporeal circulation (ecc) contributes to the morbidity and mortality. postoperative haemorrhage following ecc has been related to a platelet function defect and the activation of the blood dotting and fibrinulytic system. we investigated platelet surface antigen expression and parameters indicating activation of the clotting and fibrinolytic cascade to assess the predictive potential of these variables for increased blood loss after ecc. g0 patients referred for coronary bypass gra~ing with no history of a bleeding disorder and normal routine clotting tests were included. on the day prior to surge~ and immediately upon arrival on the intensive care unit blood samples were drawn. the surface expression of glycoprotein (gp) lib-ilia, gp lb, and p-selectin was meamred with and without in vitro stimulation with adenosine diphosphate (adp) using whole blood flow cytomet~y. platelet counts and platelet factor 4 (pf4), as well as, routine clotting tests were performed. activation of the clotting and fibrinolytic system were judged from thrombin-antithrombin-iii complex fiat), fibrinogen fig), d-dimers (dd), cc2-antiplasmin (tz2a), prothrombin fragment 1+2 (fl+2),and tissue plasm~ activator (t-pa). blood loss fxom chest tubes was measured hourly until removal of drains. following ecc the levels of pf4, tat, dd, o~2a, fl+2, and dd were sigulticnatly increased (p<0.0001) compared to baseline values. gp iib-iila, gp ib, p-selectin, platelet count, and fg were significantly reduced (p<0.0001). analysis of variance (anova) revealed that postoperative values of gp ib (p<0.0001), dd (p<o.05), fg (p<0.01),and platelet count (10<0.05) had a significant influence on blood loss. none of the preoperative data was significantly associated with increased blood loss. a subgroup of patients who bled more than the mean + 1 sd (14 cases) was formed_ in logistic regression analysis gp ib expression (p=0.01) and dd (p=0.007) were of value in identifying bleeders. the sensitivity of the equation was 56%, the stx~ificity 97% and the positive predictive value 82%. although the reasons for an increased blood loss following ecc are multifactorial, we were able to identify a small number parameters with a significant infl~nco. the combination of the expression of yon willebrand receptor on the platelet su_rfaco and d-dimer levels in plasma was successful in predicling an increased blood loss following heart surgery. this may be helpful to define indications for platelet transfusion and flesh frozen plasma. the most widely distributed receptor for the fc-region of igg is the fcreceptor iia (fcyriia). for this receptor a functionally relevant polymorphism of amino acids argmhis is known. we previously showed hit-antibodies to bind to platelets via feyriia and now whether clinical manifestation of hit is associated with the fe'/riia polymorphism. therefore, we developed a rapid two step allelle-specific pcr (asp) method for genotyping the foyriia. with control samples the asp was compared with the time consuming and complicated allele-specific oligonucleofide hybridization technique, the phenotype expression of fctriia (using the moabs iv.3 and 41h16) and the monocyte stimulation assay. complete concordance between the assays was found. we used the asp to determine the allelic distribution of the inst. physiol. chem., marburg; *)univ. of virginia, charlottesville, the functional heterogeneity of blood platelets is clinically significant and may contribute to the pathogenesis of cardiovascular and atherosclerotic diseases l). in order to understand the biochemical basis of the functional heterogeneity of platelets, we have investigated the signal transducing pathway of platelet activation a) in terms of guanylate cyclase and phosphodiesterase activities and b) at the level of protein phosphorylation and the receptor-effector coupling via g i-and gs-proteins in functional heterogeneous blood platelet subpopulations separated according to their densities. furthermore, we analyzed the adp-ribosylation of rhoa, which is a lowmolecular weight gtp-binding protein and is thought to be involved in the platelet "shape change" reaction and aggregation. aggregation was enhanced in low-density platelets (sp i) and less inhibited when cgmp was increased by no, compared to high-density platelets (spiii). sp i possessed a lower basal level of cgmp and exhibited a smaller increase in cgmp after stimulation with no. cyclic amp-dependent phosphodiesterase activity was higher in sp i compared to spiii, whereas cgmp-dependent phosphodiesterase activity was similar in sp i and sp iii. sp i, when compared to sp iii platelets, showed a higher degree of prephosphorylation of proteins in the range of 47, 32-34, and 20-22 kda. after thrombin stimulation, the phosphorylation of these proteins was similar in the subpopulations. pertussis toxin,induced adp-ribosylation of the 40-41 kda gi-protein was highest in sp i, whereas cholera toxin induced adpribosylation of the gs-protein and botulinum c3 exoenzyme-induced adpfibosylation of rhoa were highest in sp iii. the findings suggest that a) the higher aggregability in sp i compared to sp iii may be caused by a poorer ability of guanylate cyclase to form cgmp and a higher activitiy of campdependent phosphodiesterase and that b) the adp-ribosylation of g-protein subunits and rhea may contribute to the functional heterogeneity in the subpopulation leading to the greater ability of sp i for aggregation. 1) opper et al., atherosclerosis, 113, 211-217, 1995. causes of accelerated atherogenesis in non-insulin-dependent diabetes mellitus (niddm), known to be associated with increased plasma activity of plasminogen activator inhibitor type 1 (pal-l) as well as with increased plasma concentrations of proinsulin and insulin, remain enigmatic. this study was designed to determine whether proinsulin and insulin increase pal-1 in vivo thereby potentially accelerating atherogenesis. to simulate hyper(pro)insulinemia seen with niddm, we infused equimolar concentrations of insulin (1 iu/kg), proinsulin, c-peptide, or placebo, all endotoxin-free, i.v. over 1 h in 53 conscious rabbits maintained euglycemic. plasma pal-1 did not increase with placebo (n=16) or c-peptide (n=4), but did with proinsulin and insulin peaking in 3 h at 3.5-and 3.7-fold (n=16 and n=17, p<0.o02 for each) in parallel with pal-1 protein quantified by reverse fibrin autography. the increases were specific as reflected by unchanged activities of o~-antiplasmin and t-pa. pat-1 gene expression (pal-1 mrna) increased with proinsulin and insulin by 2.1-and 2.1-fold in 3 h in aorta, 1.9-and 2.4-fold in liver (p<0.05 for each), but not in heart, lung, spleen, or kidney and returned to baseline in 24 h (n=4 each). in situ hybridization localized the increased pal-1 mrna in aorta to endothelium (n=3 each). thus, both proinsulin and insulin directly augment gene expression of pal-1 in arterial endothelium in vivo, thereby increasing plasma pal-1 activity, attenuating endogenous fibrinolysis, and potentially accelerating atherogenesis. the sarco-endoplasmic-retikulum ca+÷atpases (serca) that sequester calcium into intracellular stores, represent the most important mechanism to maintain cytosolic calcium levels low and to return to a resting level after activation. recent studies in platelets and rat endothelial cells have identified two forms belonging to the serca 2b and serca 3 types. the monoclonal antibody pl/im 430 raised to human platelet intracellular membranes has been shown to recognise the serca 3 ÷+ isoform and to inhibit ca sequestration in human platelets. the aim of this study was to identify ca atpase distribution in human umbilical vein endothelial cells (huvec) and human polymorphonuclear leukocytes (pmnl). polyclonal antibodies with known specificity towards serca 2b (r 2/88) and serca 3 (89/3) in addition to pl/im 430 were used in histochemical studies of permeabilised cells and in western blotting experiments using cell lysates and mixed membranes. using the alkaline phosphatase anti-alkaline phosphatase (apaap) method r 2/88 and 89/3 reacted strongly with proteins in permeabilised huvec and pmnl, however pl/im 430 showed good staining only with pmnl. in western blotting experiments r 2/88 recognised proteins with molecular sizes of 100 kda (known to be the parent ca++atpase) and 70 kda (which may represent antibody recognised degradation product), in both huvec and pmnl. 89/3 ÷+ recognised bands at 100 kda (parent ca atpase), 74 kda and 46 kda in both huvec and pmnl membranes. in agreement with histochemical results pl/im 430 recognised proteins (100, 74 and 46 45 + + kda) only in pmnl membranes. in ca transport studies pl/im 430 ÷+ inhibited ca uptake in membranes prepared from pmnl but not in huvec. the distinct reaction of pl/im 430 with pmnl and huvec suggests the possible presence of distinct sub-isoforms of serca 3 with the pmnl and platelet proteins being similar and different to the serca 3 isoform present in huvec. microvascular endothelial cell (ec) lines with a limited life span of 2-3 months in vitro were established from human bone marrow, skin or solid tumor tissue. homogeneity of the lines was verified by flow cytometric analysis and cytokine expression patterns. the cultured ec constitutively expressed hla-class i antigens, cd44, procollagen type i, cd62e, cd34, and receptors for granulocyte colony stimulating factor (g-csf). endothelial cells constitutively produced il-6 and il-8 as well as tpa and pai-i as determined by elisa. in the presence of 5 iu/ml of exogeneous il-la, g-csf was secreted at high amounts (lng/ml) and gm-csf at low amounts (15pg/ml) in confluent monolayers and during over night incubation. these ec potently bound to isolated neutrophilic granulocytes. when granulocytes were stimulated by either 10~ 4 or 10 .7 m formyl-methionyl-leucyl-phenylanaline (fmlp) to produce oxygen radicals, the preincubation with a single cell suspension of ec caused oxygen radical scavenging. a ratio of 1 : 100 ec to neutrophilic granulocytes resulted in a 50 __+ 10% inhibition of fmlp-induced and luminol-enhanced chemiluminescence in vitro. this effect was probably due to the constitutive nitrogen oxide (no) production by ec. unexpectedly, preincubation of ec with 50 iu/ml of interferon-y (ifn-y) to increase no-secretion did not alter the extent of the oxygen scavenging effect measured at different ratios of ec to neutrophilic granulocytes. according to the surface antigen expression pattern, identical ifn-f treatment upregulated adhesion receptors on ec. concomitantly, binding of neutrophils to ec was significantly increased. thus, the net oxygen-scavenging effect by ec appears to be stable under the condition of ifn-y stimulation which indicates maintenance of ec function and effective neutralizition of non-specific oxygen radical damage. moreover, the established test system can be useful to investigate the selective contribution of other inflammatory cytokines, adhesion molecules, macrophages and platelets on ec function in vitro. atherosclerosis is characterized by intimal migration and proliferation of smooth muscle cells (smcs), by macrophage infiltration and extracellular matrix remodeling. circumstantial evidence suggests that urokinasetype plasminogen activator (upa) may be relevant in these processes. this study examines upa expression and upa antigen localization in human coronary arteries with ,carious degrees of atherosclerotic lesions. representative segments of macroscopically normal areas (mnas), early lesions (els) and fibrous plaques (fps) were processed in parallel for in situ hybddization and immunohistochemical analysis. in mnas, upa was detected both in the luminal endothelium and in celocalization with ~-actin-positive smcs throughout the tunica media. in els, upa mrna and antigen were substantially reduced in the luminal endothelium. however, cellular upa expression was detected in the subendothelial area in colocalization with tissue-infiltrating cd68positive macrophages. in the deeper intimal and in the medial layers strong upa expression was observed in association with ~-actinpositive smcs, with most of the upa-positive cells being located in areas with a high proliferative activity as detected by pcna staining. in fps, upa mrna was widely expressed in macrophage-containing areas within the fibrous cap and at the periphery of the necrotic core of the lesions. in addition, upa antigen was identified in acellular areas of the necrotic core regions. compared to the diseased intima of these lesions, the adjacent media stained only weakly for upa. in conclusion, this study demonstrates an enhanced expression and synthesis of upa by intimal smcs and macrophages in advanced atherosclerotic lesions of human coronary artedes these findings suggest a role for upa in the progression of the coronary atherosclerosis. adenosine produced by vascular cells, platelets and other tissues has been proposed to be a vasodilator, inhibits platelet aggregation, stimulates proliferation and migration of endothelial cells as well as exhibits antiinflammatory effects. adenosine of endothelial origin intra-and extracellularly formed and highly concentrated at the luminal surface may constitute an important antiaggregatory mechanism, which is part of the well-known antithrombogenicity of an intact endothelial lining. it was the aim of this study to investigate whether adenosine could influence the fibfinolytic potential of endothelial cells. when confluent human umbilical vein endothelial cells were incubated with adenosine (0.01-10mm) a significant dose dependent decrease in plasminogen activator inhibitor type-1 (pai-i) antigen production by this cells was seen as compared to control. this effect by adenosine was also time dependent and seen after 2 hours of incubation with adenosine. as determined by elisa, pai-i antigen in conditioned media of endothelial cells incubated with 10mm adenosine decreased to 57% as compared to control. pai-i antigen in extracelullar matrix of these cells was also reduced by incubation with adenosine. these results were also reflected on the level of specific mrna as determined by northern blotting. pai-i mrna decreased in endothelial cells after incubation for 4 hours with adenosine (10mm) to 77% (2.2kb) and 85% (3.2kb) of control. in conclusion our data demonstrate that adenosine can downregulate the expression of pai-1 in cultured endothelial cells thereby increasing the profibrinolytic capacity of these cells. this effect of adenosine might contribute to its antithrombotic potential in addtion to its known antiaggregatory effect. sowohl die effizienz als such die komplikationen einer antikoagulantien-dauerbehandlung sind in erster linie yon der gore und engmaschigkeit der gednnungskontrellen abh~ngig seit 1986 wird die schulung zur gerinnungs-selbstkontrolte in der herz-kreistauf-klinik bad bedebarg in gruppen eingeobt. die theoretische und praktische anleitung erfolgt in 8 stunden durch eine in der patientenschuiung erfahrene arzthelfedn (oder krankenschwested-pfleger) und einen arzt. die praktische schulung wird durch einen patientennahen theoretischen tell erg~nzt. verglichen mit konventionell ~berwachten oral antikoagulierten patienten (ca. 50% der tpz-werte liegen im theapeutischen bereich) erreichten 216 quickwert-selbstbestimmer, daf$ 83,1% yon 14 812 tpz-werten im therapeutischen bereich lagen (tabelle 1 ). schwerwiegende thromboernbolische ereignisse oder blutungskomplikatienen wurden nicht beobachtet. durchschnittlich kontrolliert der quickwert-selbstbestimmer seine gerineungswerte w6chentlich, itmgstens im zweiwochen-abstand. ob sich blutungs-und thremboembolierisiko langfristig senken lassen bei gleichze~iger verbesserung yon prognose und lebensqualit~it, bedarf des nachweises im rahmen einer randomisierten, kontrollierten und prospektiven studie. tabelle i verteilun~l der selbstbestimrnten gednnur ~swerte zeitraum 1986 zeitraum -1988 zeitraum 1986 zeitraum -1990 zeitraum 1986 zeitraum -1992 to evaluate the potential benefit of an optimized oral anticoagulation management with inr measurements performed by the patients themselves, two prospective randomized studies have been completed. the first study (n=60) was performed to evaluate the accuracy of inr self-testing (inr-st) compared with standard laboratory controls as well as the improvement of anticoagutation monitoring by inr-st, e.g. the percentage of measurements found inside the target therapeutic inr-range. the 231 parallel measurements revealed a high accuracy of inr-st with a 4.5% median difference between parallel controls of single measurements. in addition, inr-st resulted in a significant increase of measurements inside the target therapeutic range (from 54 to 80%) in the entire group. however, inr-st performed every 4 weeks did not result in a significant improvement, while selfcontrols every week (from 44 to 84%) and every 2 days (from 51 to 84%) did. in a second study (150 patients) the incidence of bleeding and thromboembolic complications were evaluated in patients who either had standard anticoagulation measurements o[ performed inr-st. during a median follow-up of 1.5 years in the group with standard oral anticoagulation management an incidence for bleedings of 10.9% per year and for thromboembolic complications of 3.6% per year was found, while in the group of patients with inr-st bteedings occured with an incidence of 4.5% per year and thromboembolisms with 0.9% per year. it can be concluded that inr-st can be performed with high accuracy by the patients themselves. inr-st results in more frequent controls of the inr and consequently in significantly longer time periods where the patients are inside the optimal target therapeutic inr range. the more accurate anticoagulation management resulted in our study group in a significantly lower incidence of thromboembolic and hemorrhagic complications. the cell biology of tissue factor (thromboplastin) h. prydz, the biotechnology centre of oslo, norway tissue factor (tf,thromboplastin) is the most potent trigger of blood clotting knuwn.lts presence in tissue extracts has been known since before the turn of the century.erwin chargaff (of dna fame) first described that tf consisted of two parts (a protein and a lipid moiety), a fact that was independently rediscovered in the early sixties by deutsch and lechner in vienna and by myself in oslo. in 1973 tf was first purified and shown to be an integral membrane protein of mr about 50 kda (this was a slight overestimate,recent studies indicate 46-48 kda). in 1987 four groupes independently cloned tf edna, later also genomic dna. the structure of tf suggested that it was a membrane spanning receptor, with an extracellular part of about 219 amino acids folding into two domains remotely like the domains of the ig-superfamiliy, and more specifically the eytokine receptors. tf is synthesized to a varying degree in many tissues, but not normally in tissues in contact with blood. however, during acute inflammatory situations and under the influence of a number of agents (immune cnmplexcs, lectins, cytuklnes,bacterial components, complement components etc) monucytes and vascular endothelial cells are induced to synthesize tf. another possible way tu get tf into contact with blood is thruugh trauma, surgical ur otherwise. the fact that tf is inducible has created a lot uf interest in its cell biology. we reported recently that binding of factor viia tu tf un the cell surface elicited a marked increase in intracenular ca spikes, thus rendering the final proof that tf not only luoks like a receptor but really is one, since all the three essential criteria are fulfilled: tf has a transmembrane segment and a cytoplasmic tail tf has a high specificity/high affinity ligaud (factor vii and factor x) tf induces an intracellular signal upon ligand binding recent results regarding tf as a receptor and regarding its intracellular traffic will be reported. both f.ix and f.x are activated by the tf/f.viia complex. we have used a tf-initiated model system to examine the roles played by f.ixa and f.xa in initiating coagulation. the in vitro model contained a cellular tf source; plasma concentrations of f.ii, v, viii, ix and x, tissue factor pathway inhibitor and antithrombin iii. we tested the ability of exogenously added f.ixa or f.xa to substitute for tf/viia activity in initiating platelet activation and thrombin generation. f.xa was 10 times more potent than f.ixa in initiating platelet activation, but f.ixa was more potent in promoting iia generation. in the presence of tf/viia, zymogen f.x was required both for platelet activation and iia generation, while f.ix was only required for iia generation. thus, tf/viia-activated f.ixa and f.xa have distinct physiological roles. the main role of f.xa is to activate platelets by producing an initial small amount of iia. the process of platelet activation is much more efficient if this initial iia is produced on the tf-bearing surface. f.ixa, by contrast, enhances iia generation on platelets by providing f.xa for platelet surface prothrombinase assembly. we conclude that activation of both f.ix and f.x by tf/viia is essential for efficient initiation of coagulation. furthermore, f.x activated on the surface of a tf-bearing cell isnot functionally equi.valent to f.x activated on the platelet surface. therefore, f.x activation by tf/viia does not compensate for a lack of f.ix or viii in hemophilia because the f.xa activated by tf/viia is located on the wrong surface for efficient thrombin generation. our results emphasize that the location, of an activated coagulation factor is a critical determinant of its role in hemostasis. the x-ray crystallographic structure of the fviia-tf complex located the areas within fviia in contact with tf to the first egflike and the protease domains. high-affinity binding of fviia to tf depends on ca2+ and previous studies suggest that ca2+ binding to a site in the protease domain is required. however, using surface plasrnon resonance we have shown that removal of the n-terminal gla domain and hydrophobic stack (hs) from fviia results in a decreased affinity for tf. in search for the mechanism by which regions in fvua not in direct contact with tf affect the affinity, various techniques have been employed. a plausible model is one in which the gla domain and hs optimize the interaction with tf by affecting regions that mediate the association with tf. using gel filtration we demonstrated that the hydrodynamic radii of des(1-38)-fviia and, especially, fviia decreased upon ca2+ binding, whereas des(1-44)-fviia was virtually unaffected. this suggested that the gla domain and the hs interact in a ca2+-dependent manner with other regions in fviia, presumably the protease domain, making fviia more globular. using cd spectroscopy, we could detect ca2+-induced changes in the secondary structure of fviia, all assignable to an increased helical content of the gla domain. the amidolytic activity of fviia in the absence of tf has a ca2+ dependency that suggests involvement of the gla domain in obtaining full activity. this activity was very sensitive to guanidine hydrochloride (c50<0.5 m) and its quenching corresponded to unfolding of the ca2+-loaded gla domain as measured by changes in the trp fluorescence of fviia. based on these results it is conceivable that the gla domain and perhaps the hs of fviia interacts with the protease domain and that this globular form mediates the initial contact with "it. several studies have indicated a profound role for fvii(a) in arterial thrombogenesis. in the present study we quantified the inhibitory effect of dansyl-glutamyl-glycyl-arginyl-chloromethyl ketone recombinant fviia (degr-rfviia) on acute thrombus formation at medium-sized artery blood flow conditions characterized by a wall shear rate of 650 s -1. native human blood was drawn directly from an antecubital vein by a pump into a heparin coated mixing device where degr-rfviia (0.9 -880 nmol/l final plasma concentration) or buffer were mixed homogeneously with the flowing blood. subsequently, the blood entered a parallel-plate perfusion chamber in which a thrombogenic surface consisting of immobilized tf and phospholipids was positioned. fibrin deposition, platelet-fibrin adhesion and platetet thrombus volume triggered by this surface were measured by morphometry. degr-rfviia inhibited these thrombus parameters in a dose dependent manner with ic50 values between 0.5 and 0.7 nmol/l the plasma levels of thrombin-antithrombin m complexes were inhibited at an ic50 of 0.6 nmot/l since the concentration of fvii and fviia in normal plasma is about 10 and 0.1 nmol/l, respectively, theic50 values indicate that degr-rfviia is a very efficient inhibitor of thrombus formation in this model. thus, inhibition of the extrinsic pathway of coagulation may represent and entirely new and effective arterial anti-thrombotic approach. the anticoagulant agent from medicinal leeches, hirudin, is presently gaining popularity as an antithrombotic drug. the design of recombinant forms of the minipretein and their availability led to the clinical use of this naturally occurring thrombin inhibitor. extensive studies in experimental animals have shown that due to its pronounced and specific antithrombin effect hirudin is an antithrombotic agent of high quality. apart from its anticoagulant effect, hirudin is pharmacodynamically inert and is well tolerated in vivo. brain abnormalities in male children and adoleescents with hemophilia: detection with mr imaging mri of sickle cell cerebral infarction in addition, portions of the preparations were incubated with 1.4nm-gold labelled fibrinegen molecules (fg-au; final concentration 4gp_g/ml) for 10 see at 37°c. the expenments were stopped al~er 3 or 10 rain by glutaraldehyde fixation. serial sections were prepared and examined after silver-enhancement using danscher's (1981) method to visualize the fg-au. hwdid not alter the resting gfp. after .3,rain, activated hw-platelets changed their shape moderately but in contrast to the controls did not aggregate. moreover, the conatdcting cytoskeleton that centralizes platelet organelles was not formed. fg-au was found on the surface of the control platelets, in high density in the contact spaces between the aggregated platelets and internalized in the surface connected system. only a very small number of hwtreated cells had fg-au on their surface and showed internalization. 10 rain after adp most of the platelets in both preparations had retumod to a discospherecytic shape it is confirmed that hw inhibits the platetet aggregation and in addition, 1) the formation of the contractile cytnskeleton as well as 2) the binding and interalization of the used ligand fibrinogen santoso 2 l.institute for immunology and transfusion medicine, emst-moritz-arndt-university greifswald; 2. institute for transplant vasculopathy in cardiac allografts is the leading limitation to long-term survival of cardiac transplant recipients. activation of the coagulation mechanism by adherent monocytes expressing tissue factor (tf) is expected to be involved in the development of transplant atherosclerosis. in this in vitro study the effect of the immunosuppressant cyclosporine a (csa) on monocyte tf expression and the regulation of the tf gene transcription was analysed. csa was shown to inhibit the induction of monocyte procoagulant activity (pca) by decreasing lipopolysaccharide (lps)-induced tissue factor (tf) expression in monocytes. csa exerted a dosedependent inhibitory effect on monocyte tf expression at concentrations (1,5 txmol/l csa: 50 % inhibition) not decreasing the cellular protein synthetic rate. addition of csa during lpsstimulation prevented activation of the nuclear factor (nf) ~b as demonstrated by electrophoretic mobility shift assay. western blot analysis confirmed reduced nuclear translocation of nf-~b in the presence of csa. inhibition of the lps-induced nf-~b activation by csa resulted in a reduced tf gene transcription as demonstrated by rt-pcr-analysis. thus, cyclosporine a prevents tf expression by inhibiting the induction of tf gene transcription in activated monocytes. csa may therefore provide a therapeutical tool acting not only as a direct immunomodulating agent, but also by influencing local coagulation activation. the n-glyean pattern of recombinant human coagulation factors ii (rf-i~ and ix (rf-ix), derived from both transfected chinese hamster ovary (cho) cells and african green monkey (vero) cells, and produced at industrial scale were analyzed by binding to carbohydrate specific lectins and were compared with the glycan structure of human plasma-derived coagulation factors. human plasma-derived coagulation factors 1i (hpf-i1) and ix (hpf-ix) exhibited complex-type glycan structures with carbohydrate chains capped with c~(2-6)sialic acid. terminal galactose-b(1-4)-n-acetylglucosamine units were detected in hpf-ix. both cho cell-derived rf-]i and rf-ix exhibited complot-type glycosylafion and contained ~2-3)-sialio acid in addition to terminal galactose-b(1-4)-n-acetylglucosamine. vero cell-derived if-ix exhibited a complex-type glycan structure sinailar to that of cho cell derived rf-ix. in contrast, rf-li produced by vero cells exhibited a glycan microheterogeneity composed of hybrid-type glycosylation containing "highmarmose" structures and complex-type glycosylation containing et(2-3)-sialie acid. galaetose-b(1-4)-n-acetylglucosamine structures and a low concentration of ct(2-6)-sialic acid were detected in both micro-heterogeneity fractions of veto cell-derived rf-li. although different in the'tr carbohydrate structures, coagulation factors ii and ix obtained recombinantly from both transformed cho-cells and vero-cells exhibited coagulation activities comparable to the plasma-derived proteins. exposure of tissue factor (tf) to flowing blood, as a consequence of vascular injury, leads to tf/f.viia-initiated coagulation which may play a key role in triggering intravascular thrombosis. in order to evaluate the potential advantages of tf pathway blockade over existing therapy, the antithrombotic and the anticoagulant .effects of a monoclonal anti-rabbit tf antibody (ap-1) and heparin were compared in a rabbit arterial thrombosis model. thrombosis was induced by a local injury to the carotid artery and the recurrence of thrombus formation was followed by monitoring the cyclic flow variations (cfv) of the arterial blood flow. rabbits received intravenously either ap-1, heparin or vehicle, and cfv were monitored on line for 120 min. activated partial thromboplastin time (aptt), and activated clotting time (act) were measured at the end of the experiment control ap-i heparin <lmg/kg* 0.2mg/kg/hr 0.8mg/kg/hr intraabdominallretroperitoneal, cns) on a named-patient basis. later also mild/moderate bleeds, predominantly joint bleeds were included.in 80-90% of the bleeds novoseven was found to be hemostatically efficacious. also in major surgery (hip-and knee-replacement including a bilateral total condylar arthroplasty, herniotomy) hemostatic efficacy has been found to be 94% since rfvlla is proteotytically active only in complex with tf the risk for systemic activation of the coagulation system should be minimal. also very few side-effects have been seen.doses between 75-120 tjg/kg have been used, which initially have to be given at 2 hours intervals. to achieve satisfactory hemostasis both an initial dose high enough to induce immediate hemostasis and an appropriate dose interval ensuring a plasma level of fvli:c well above the hemostatic lower level for a period of time, the length of which is dependent on the type of bleeding, are important. accordingly, the highel the initial dose the more robust with regard to dose interval will the treatment be. acute myocardial ischemia is usually triggered by coronary thrombosis. heparin (hep), along with aspirin, is commonly used for the treatment of unstable angina (ua) and myocardial infarction (mi). hirudin (hiq is a potent antitbrombin agent which may offer therapeutic advantages over standard hep for the treatment ua and mi. the oasis phase i pilot study conducted in 24 canadian centres, has evaluated the safety, feasibility and efficacy of hir (hbw 023, behringwerke ag, marburg) compared to hep in 601 patients with mi or suspected non q-wave mi. patients (95 % received aspirin) were randomized to a 72 hr infusion of either hep (5000 u bolus followed by 1200 u/hr; n=253), or low dose hir (0.2 mg/kg bolus then 0.10 mg/kg/hr; n=175) or medium dose lair (0.4 ms/ks bolus then 0.15 mg/kg/hr; n=173). at seven days of follow-up the incidence of major bleeds was 0.4 % in the heparin group, 0.6 % low dose hit, 1.2 % medium dose lair (p=ns), minor bleeds were 10.3 %, 17.1% and 19.1% respectively (p---0.008 hep vs med dose); there were no hemorrhagic strokes. the incidence of the primary outcome of cardiovascular death, new mi and severe recurrent ischemia (refractory or severe angina) was 17.0 %, 15.4 % and 8.7 % respectively (p---0.014 hep vs reed dose). there was also a trend towards lower rates of coronary artery bypass surgery in reed dose hir compared to hep. in a 200 patient substudy, levels of thrombin antithrombin complex, fibfinopeptide a and d-dimer were all suppressed to a greater extent by mr compared to hep. these results indicate that hirudin may be a more effective agent for the treatment of acute coronary syndromes, but more information on safety and efficacy is needed from larger randomized trials. full clinical and coagulation resuks will be presented. hat is caused by an immunologic response to heparin. affected patients need effective parenteral anticoagulation. we treated 82 patients (30m,52fm), median age 61 years (18-84) with laboratory confirmed hat (hipa-test) with r-hirudin (behringwcrke ag, marburg). treatment regimens were: ai acute hat: 0.4 mg/kg b.w., continuous infusion 0a5 mg/kg b.w/h (51 patients); a2 acute hat with thrombolysis: bolus 0.2 mg/kg b.w., continuous infusion 0.1 mg/kg b.w./h (5 patients); b: proph, treatment: continuous infusion 0. i mg/kg b.w.pa (18 patients); c: during cardio-pulmunary bypass. primary objective was: increase in platelet counts or maintenance of normal baseline values during effective anticoagulation. secondary objectives were the incidence of new thromboembolic complications (tecs), major hemorrhage, limb amputations and deaths. results were compared with a historical control of 120 ix-at patients, all confirmed by the hipa test, but treated before rhirudin was available (danaparoid, marcumar, no anticoagulation). primary efficacy analysis: respunders ai: 74%o, a2: 60%, b: 44%, c: 50%, total: 65%. all adverse events beside bleeding complications had been judged to be caused by the underlying disease and not directly related to r-hirndin. proportion of patients with event in the obse~wation period; death: a1 5.9%, a2 0%, b 16.774, c 0%, total: 7.3%, during rhirudin treatment: 1.2%; new tec: ai 5.9%, a2 0%, b 27.8%, c 0%, total 9.8%, during rhirudin: 2.4%; limb amputation: ai 3.9%, a2 0%, b 5.6°/0, c 0%, total: 3.7%, during rbirudin: 3.7% eaapoiat we conclude, that r-hirndin is an effective anticoagulant in hat typa ii patients allowing rapid re, coved" of platelet counts. compared to a historical control the r-hirudin group had a strongly reduced incidence of combined endpuint of mortality, limb amputation and new tecs. major bleedings occurred slightly more frequent than in the historical control. however, the dosage used in regimen b might have been too low, as flds group revealed an effective anticoagulatiun and increase in platelet counts in only 43% and concomitantly had the higl)est incidence of new tecs. key: cord-031907-ilhr3iu5 authors: nan title: isev2020 abstract book date: 2020-07-15 journal: nan doi: 10.1080/20013078.2020.1784511 sha: doc_id: 31907 cord_uid: ilhr3iu5 nan introduction: primary tumours secrete large amounts of extracellular vesicles (evs), which play critical roles in preparing distant sites for a pre-metastatic niche formation, thereby promoting metastasis and even determining metastatic organotropism. whether biogenesis, secretion rates and organotropism of evs are linked remains unknown. we have recently shown that ral gtpases control evs secretion in nematodes as well as in mouse mammary tumour cells (hyenne et al. jcb 2015) . since both rala and ralb are overexpressed or over-activated in various human cancers, we aimed to investigate the mechanisms by which these two gtpases control evs secretion and to determine how this affects metastatic progression, with a focus on breast cancer. methods: we used 4t1 mouse mammary carcinoma cells knocked down for either rala or ralb and determined their ability to induce orthotopic tumours and metastasis in a syngeneic mouse model. in vitro, we investigated ev secretion mechanisms using confocal and electron microscopy (em). evs were isolated either by uc or sec and characterized by nta, em, rna sequencing and mass spectrometry. the function of evs was assessed using a transwell assay. finally, we tracked the organotropism of fluorescently labelled evs and their capacity to induce pre-metastatic niches in mice. results: we show that rala and ralb promote lung metastasis of breast cancer cells in mice without affecting their invasive behaviours. we found that rala and ralb control the biogenesis of exosomes, by acting on the formation of multi-vesicular bodies though the phospholipase pld1. as a consequence, knock down of rala or ralb reduces the levels of secreted evs and modifies their rna and protein contents. these differences alter the pro-tumoural function of evs, as demonstrated with an in vitro permeability test. importantly, we show in vivo that evs from rala or ralb depleted cells have a decreased lung organotropism and, as a consequence, are less efficient in priming lung metastasis. finally, we show that high expression of rala or ralb is associated with a bad prognosis in human breast cancer patients. summary/conclusion: altogether, our study identifies ral gtpases as central molecules linking the mechanisms of evs secretion, their dissemination and their capacity to promote metastasis. nuclear proteins are recruited into tumour-derived extracellular vesicles upon expression of tetraspanin tspan8 introduction: tetraspanin tspan8 is a transmembrane protein that exhibits a unique expression pattern, being overexpressed in many cancer types, but undetectable in most healthy tissues. although there is increasing evidence of an effect of tspan8 in invasion, metastasis, and regulation of extracellular vesicle cargo, the molecular mechanisms of tspan8 are yet not fully understood. methods: to study the function of tspan8, we have established a fibrosarcoma model consisting of the parental cell line (ht1080) and its derivatives expressing tspan8 (ht1080-tspan8) either fused with different fluorescent tags or tag-free. life imaging, sted and storm microscopy were used to determine the intracellular localization of tspan8. co-immunoprecipitation from nuclei lysates was performed to detect direct and indirect interacting partners of tspan8. small evs were purified from cell-conditioned media using sec and subjected to mass spectroscopy and ngs for a comprehensive comparative analysis of the proteome and transcriptome of the evs. results: the results of the proteome analysis showed a strong effect in the protein cargo of evs upon tspan8 expression. remarkably, among 20 of the most regulated targets, several histones and ribosomal proteins were enriched in the evs derived from ht1080-tspan8 cells. in line with this finding, life imaging and super-resolution microscopy revealed that, while a majority of the intracellular tspan8 is located on the cell membrane or intracellular membranes, -as it is known for other tetraspanins-, a portion of tspan8 is located on the nuclear envelope. in fact, several histones co-immunoprecipitated with tspan8, indicating their interaction. summary/conclusion: our data show that the expression of tspan8 in the tumour cells greatly impacts ev cargo. moreover, localization of tspan8 on the nuclear envelope, together with the enhanced recruitment of nuclear and ribosomal proteins to the evs, suggests a new mechanism of action of tspan8. introduction: extracellular vesicles (evs) modulate tissue development, regeneration and disease through the transfer of proteins, nucleic acids and lipids between cells. currently, the mechanism of cytosolic delivery of ev cargo is largely unknown. here, we unravel how evs release their cargo in recipient cells. methods: evs were isolated from gfp-cd63 and cd63-rfp expressing hek293 t cells by ultracentrifugation. gfp-cd63 and cd63-rfp evs were added to hek293 t cells stably expressing anti-gfp fluobody and fluorescently tagged galectin-3, respectively. clem and fluorescence microscopy were employed to visualize fluorescent markers in recipient cells. bafilomycin a1 and u18666a were used to inhibit endosomal acidification and cholesterol export from lysosomes, respectively. results: fluorescent galectin-3 which binds to betagalactosides present at the luminal side of endosomes was used to detect endosomal permeabilization. the absence of galectin-3 recruitment to endosomes in presence of cd63-rfp evs showed that endosome permeabilization is not the mechanism behind ev cargo release. gfp-cd63 ev addition to cells expressing anti-gfp fluobodies resulted in the formation of fluobody punctae, reflecting cytosolic exposure of ev cargo. subsequent clem of the fluobody punctae revealed endosomes as the underlying cellular compartments from where cargo release takes place. neutralization of endosomal ph and accumulation of endosomal cholesterol blocked cargo release, showing that ev cargo release is dependent on endosomal ph and cholesterol level. summary/conclusion: we show that genetically encoded cytosolic probes and clem offer an excellent approach to study both the mechanism and efficiency of ev cargo release in cells. we provide experimental evidence that ev cargo release occurs from endosomes. funding: the research was supported by dutch technology foundation ttw and netherlands organization for scientific research nwo, de cock-hadders stichting, and erasmus mundus namaste scholarship. healthy humans. to determine cut-off values for diagnoses, reference intervals of evs in plasma are needed. to establish such reference intervals, (1) a significant number of healthy donors should be included, (2) the presence of non-ev particles, residual platelets, lipoproteins, and haemolysis should be quantified, (3) flow cytometry signals should be in si units. the long-term aim of this study is to determine reference intervals of ev concentrations in human plasma within known dynamic ranges of the detectors. methods: (1) to establish a clinical reference, we collected blood from 224 healthy volunteers and prepared platelet-free plasma. (2) we performed quality control measurements including residual platelet count, serum index, and lipid spectrum. (3) we measured all samples by flow cytometry (apogee a60-micro) and used custom software (matlab r2018b) to automate calibration of all signals and data processing. scatter signals were calibrated in comparable units of scattering crosssection (nm2) and diameter (nm). fluorescence signals were calibrated in units of molecules of equivalent soluble fluorophores (mesf). results: the quality controls showed that most residual platelet concentrations ranged from 10^5 to 10^6 per ml except for one outlier, while the serum index and lipid spectrum were normally distributed. preliminary results of the first 21 donors analysed, show that within the ev size range of 162-1,000 nm, the median concentration of cd61+ evs is 3.9•10^8 per ml (apc>150 mesf), cd62p+ evs is 1.1•10^7 per ml (pe>83 mesf), cd235a+ evs is 5.2•10^7 per ml (pe>123 mesf), and cd45+ evs is 1.8•10^7 per ml (apc>91 mesf). summary/conclusion: we have developed reliable procedures for establishing reference intervals of ev concentrations, within a well-defined size and fluorescence intensity range, in human plasma by flow cytometry. we are currently applying these procedures to 224 samples to obtain, for the first time, ev reference intervals for human plasma. funding: pol, e. van der is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni 15924. introduction: the use of extracellular vesicles for diagnostic and therapeutic applications has seen a major interest increase in recent years because of their capacity to exchange components such as nucleic acids, lipids and proteins between cells. isolation of a pure population of evs is the first step in studying their physiological functions since contamination of ev preparations with non-ev proteins can lead to incorrect conclusions about their biological activities. we have developed a new method termed tangential flow for analyte capture (tfac) using ultrathin nanomembranes to purify extracellular vesicles from pure, highly complex biological fluids such as blood plasma, resulting in a new method for extracellular vesicle purification. methods: the tff microfluidic devices are assembled through a layer stack process using patterned polydimethylsiloxane (pdms) sheets with the membranes sandwiched between top and bottom channels. undiluted plasma was tested in both normal flow filtration (nff) and tangential flow filtration (tff) modes on ultrathin nanomembranes. we have utilized a pore patterning technique called nanosphere lithography (nsl) that uses close-packing of nanoscale beads to pattern pores in an ultrathin membrane. results: nff of undiluted plasma resulted in a protein cake of~8 μm on the membrane, which prevented further transport across the membrane and evs were buried in the formed cake that were impossible to identify. however, tfac as a modified version of tff, led to capturing cd63 positive evs on the pores of the membrane with little evidence of protein fouling. nsl allows us to fabricate nanopockets (bowls with a single pore at the base) with various diameter, depth and pore diameter. using nsl, we further utilize nanopocket membranes to purify ev samples in tfac devices. this nanomanufacturing technology will allow us to pattern nanopockets with various diameter, depth and pore diameter which increases the efficiency of capturing of evs. furthermore, nanopockets can be modified and coated by specific ev markers to capture different subpopulation of evs based on size and affinity and further allows identifying the phenotypic subsets of evs by combining both size and affinity-based techniques. summary/conclusion: we have developed a method for the capture and release of nanoparticles such as evs called tfac using ultrathin nanomembranes. nsl technology can be applied to fabricate nanopockets with different physical and biochemical properties. utilizing nanopocket membranes in tfac system will allow us to separate different subpopulations of evs based on size and affinity. funding: this project was supported in part by the national science foundation (iip 1660177) to j.l.m and t.r.g., department of defence (ca170373) to j. l.m., and the national institutes of health (r35gm119623) to t.r.g. the addition of a size exclusion chromatography step to various urinary extracellular vesicle concentrating methods reveals differences in the small rna profile introduction: urinary extracellular vesicles (evs) and their rna cargo are a novel source of biomarkers for various diseases, however non-vesicular rna (e.g. associated with proteins) is also present within urine. this study aimed to identify the optimal method for isolating and enriching evs from human urine prior to small rna analysis. methods: three ev concentration methods, ultracentrifugation (uc); a precipitation-based kit (pk); and ultrafiltration (uf), were compared using 50 ml aliquots of pooled healthy volunteer urine. evs were then separated from protein contaminants by size-exclusion chromatography (sec). presence of evs was confirmed by transmission electron microscopy and western blotting, and evs were quantified using nanoparticle tracking analysis (nta). small rna content of concentrated urine and fractions obtained by sec (evs and proteins) were evaluated with the agilent bioanalyzer small rna chip. results: ev recovery following sec of concentrated samples was 35-78%, however particle: protein ratio (indicating ev purity) was approximately 10x greater after sec, regardless of the concentrating method used. uf+sec yielded the highest number of evs (per ml of urine) compared with pk+sec and uc+sec. small rna analysis from uf-concentrated urine (prior to sec treatment) identified peaks at 20 nucleotides (nt) and 60 nt. following sec, rna analysis indicated that ev fractions contained mostly small rna of~60 nt, whereas the protein factions contained small rna of 20 nt in size (consistent with mirnas). summary/conclusion: uf+sec provided the best balance between ev recovery (per ml urine) and particle: protein ratio. these data indicate that most of the 20 nt sized rnas, presumably mirnas, are not within evs in urine. ev preparations obtained after uc, pk and sec (regardless of concentrating method) contain pre-dominantly~60 nt sized small rna. these data outline the importance of removing non-vesicular proteins and rna from urine ev preparations prior to small rna analysis. funding: this research has been funded by petplan charitable trust. the use of rev for the optimization of ev separation and characterization by af4 introduction: the reproducibility of extracellular vesicle (ev) research has been hampered by the infinite number of separation and measurement techniques and the lack of appropriate reference materials (van deun et al., nat methods, 2017) . recombinant extracellular vesicles (rev) were developed as a biological reference material to overcome these limitations (geeurickx et al. nat comm 2019) . since rev have ev-like physical an biochemical characteristics and as they are trackable and distinguishable from sample ev they can be used as a spike-in material for data normalization and method development, and as a quality control. we used rev to optimize ev separation by asymmetrical flow field-flow fractionation (af4). methods: an af4 long channel column with a frit inlet driven by the eclipse system (wyatt) was coupled to a uv detector (shimadzu), mals dawn helios-ii (wyatt) and fluorescent detector (agilent). a spacer of 350 µm and a regenerated cellulose membrane of 10 kda were used. pbs supplemented with 0.02% nan3 was used as a running buffer. light scatter profiles and uv profiles were analysed as well as the fluorescent emission spectrum as the rev are gfp positive. fractions were collected and analysed by nanoparticle tracking analysis (nta) and western blot. we also estimated the repeatability and reproducibility of the af4 technique by light scatter and fluorescence profiles as well as the recovery efficiency by nta. results: in a first step 4*10^10 rev isolated from conditioned medium by a velocity gradient were injected in the af4 system to optimize the ev characterization protocol. later concentrated conditioned medium was spiked with 1*10^11 rev and injected in the af4 column to optimize ev separation from non-ev contaminants. the most optimal separation protocol was obtained by varying detector and cross-flow settings. this protocol shows elution of monodisperse particles at each time point and size distribution estimations by af4 correspond to size determination by nta and electron microscopy. summary/conclusion: we were able to optimize the af4 protocol for characterization of ev by af4 as well as for separation of ev from crude conditioned medium samples by using rev. we demonstrate that rev are suitable for method development and that af4 has high potential as an ev separation technique. comparative evaluation of ev isolation methods for ev subpopulation analysis in human urine, plasma and cell culture media liang dong, richard zieren, kengo horie, sarah amend and kenneth pienta the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: extracellular vesicles (evs) are membrane-enclosed particles of variable sizes that are released by any cell types to the extracellular space and are identified in all body fluids. a shortcoming in ev research is the lack of standardized isolation protocol for various sample types, resulting in heterogeneous outcomes in downstream analyses. in this study, we compared the ev isolation purity and efficiency among ultracentrifugation (uc), precipitation, sizeexclusion chromatography (sec) and a microfluidic tangential flow filtration device (exodisc) in human plasma, urine and cell culture media (ccm). methods: all evs were isolated by different isolation methods and characterized per misev2018 guidelines. single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence and nanoflow (nfcm) were used for single particle analysis. results: in ccm, total particle yield of exodisc was about 5 times higher than those of the rest three methods. size distribution differed per sample, but the ranges were comparable between the different isolation methods. the total protein amount of sec, precipitation and exodisc were similar which were 6-10 times higher than that of uc. uc had the highest particle-toprotein ratio followed by exodisc. precipitation and sec had low ratios. when loading 9ug of total protein for western blot, cd9, cd81, cd63 and flot1 could only be detected in uc and exodisc samples, but not precipitation or sec. sp-iris and nfcm demonstrated consistent purity findings. in urine, total particle yields of exodisc and sec were about 4 times higher than those of the rest two methods. the total protein amount of precipitation was 3 times higher than exodisc and sec, 10 times higher than uc. sec had the highest particle-to-protein ratio followed by uc and exodisc. precipitation had low ratios. in plasma, total particle yields of exodisc and precipitation were 100 times higher than those of the rest two methods. and so were the total protein amount. sec had the highest particle-to-protein ratio followed by uc. exodisc and precipitation had low ratios. western blot, sp-iris and nfcm demonstrated consistent purity findings in urine and plasma. to evaluate particle capture efficiency, we spiked a known number of density-gradient uc purified evs to each method and the recovery rate of uc, precipitation, exodisc and sec was 8.6%, 31%, 50.1% and 42%, respectively. summary/conclusion: the order of ev isolation purity in ccm is uc, exodisc, sec and precipitation. in urine it's sec, exodisc, uc and precipitation. and in plasma, this order is sec, uc, exodisc and precipitation. exodisc and sec have similar high isolation efficiency followed by precipitation. uc has low efficiency for ev capture. a capillary-channelled polymer (c-cp) fibre spin-down tip approach for the isolation and biomarker characterization of extracellular vesicles of ovarian cancer origin kaylan d. kelsey, rhonda r. powell, terri f. bruce and r. kenneth marcus clemson university, clemson, usa introduction: extracellular vesicle (evs) profiling has shown promise for disease detection through less invasive sampling (liquid biopsies). current diagnostic tools for ovarian cancer are invasive or only semiinformative. thus, use of evs could prove useful in early disease detection. demonstrated is a hydrophobic interaction chromatography (hic)-based capillarychannelled polymer (c-cp) fibre tip spin-down process for the isolation of ovarian cancer evs for use in diagnostics. methods: polyester c-cp fibre micropipette tips are employed in the isolation of evs from biological matrices including cell culture media, urine, and blood plasma in a spin-down solid-phase extraction (spe) approach. evs were isolated from standards of healthy urine origin and from skov3 cells (human ovary adenocarcinoma). the c-cp fibre isolation method (taking less than 5 mins and 10 μl sample volumes) preserves the morphology and functionality of evs as confirmed by sem, tem, and confocal fluorescence microscopy. results: the dynamic binding capacity of ev standards on a 1 cm pet c-cp fibre tip was found to be~7e11 particles (50%). the release of evs was confirmed using dot blot analysis for cd9, cd81, and cd63 tetraspanin proteins. immobilized evs were subjected to immunolabeling to allow the positive identification of a profile of ovarian cancer biomarker proteins (her2, cd24, egfr, epcam, ca125). summary/conclusion: this new ev isolation method introduces a simple capture mode, allowing for direct immuno-characterization and imaging on the fibre surface. this offers a unique and cost-effective opportunity for clinical analyses related to early detection and diagnosis of ovarian cancers (and others). the longterm goal is the creation of a rapid ev isolation and biomarker detection platform. funding: support from the national science foundation, eppley foundation for scientific research, gibson foundation, prisma health system and itor biorepository are gratefully acknowledged. development and optimization of purification method of exosomes by tangential flow filtration and ion-exchange chromatography approach tek lamichhane, ali navaei, sandeep choudhary, yonatan levinson and senthil ramaswamy cell & gene therapy r&d, lonza inc, rockville, usa introduction: extracellular vesicles (evs) such as exosomes have significant therapeutic potential, however, translation of ev-based therapies has been slowed down because of the biomanufacturing challenges. the isolation of evs, especially exosomes, is inherently challenging due to their small size, and heterogeneity in the mixture. the current isolation methods either have low recovery rate, aggregation, damaging the structure, time consuming or co-precipitation of contaminants. specially, it is difficult to process larger sized samples by centrifugation-based or immunoaffinity based methods because of the time and cost associated with these methods. methods: to overcome these roadblocks, we developed and optimized alternative purification techniques to isolate evs with higher purity and yield by using tangential flow filtration (tff) coupled with ion-exchange chromatography. we used bioreactor platform to produce evs from serum-free medium using bm-msc and hek293 s cells. bm-mscs were cultured on stirred tank bioreactors using microcarriers which provide a high surface area to volume ratio for the optimal cell growth and evs production. impellers were used to enhance mixing and maintain homogeneous culture conditions that can be easily monitored and controlled. results: depth filtration was applied for clarification of conditioned medium. we screened different types of filters during depth filtration for the best recovery of evs. tff membranes with different pore sizes were used to optimize the purity and yield of evs. because of the negatively charged nature of evs, anion exchange chromatography was chosen to capture and separate tff purified vesicles by their surface charge characteristics. we compared monolith based and membrane-based anion exchange columns to remove contaminants and purify exosomal fractions. the purity, size and presence of exosomal markers in isolated evs at each step of purification was evaluated by f-nta, nano-fcm and tetraspanins based elisa kits. summary/conclusion: in summary, our optimized methods improved the speed of isolation and purity of evs to the clinical grade. the production and isolation methods of exosomes that we developed here will be easily expandable to support large-scale and cgmp compatible bio-manufacturing in the future. use of an alternating current electrokinetic microelectrode chip to positively identify oncology, neurology, and infectious disease samples through plasma extracellular vesicle analysis juan pablo hinestrosa, jean lewis, david searson, orlando perrera, alfred kinana, heath balcer and rajaram krishnan biological dynamics, inc., san diego, usa introduction: cancer, neurological, and infectious diseases are leading causes of death, with early detection needed to improve outcomes. extracellular vesicles (evs) in the blood contain disease biomarkers, but current methods do not allow rapid analysis, and are often limited to one biomarker type. methods: we developed methods using alternating current electrokinetics (ace) to isolate evs from bloodbased samples and analyse the evs in situ with downstream assays for protein and nucleic acid biomarkers. we investigated if we could identify tuberculosis (tb) donor samples, protein and nucleic acid biomarkers in evs derived from cancer cell lines, and alzheimer's disease (ad) protein biomarker levels. results: ev isolation was confirmed by positive identification of the proteins cd9, cd63, and cd81 and measurement of ev mrnas using a direct rt-ddpcr assay. different disease models were analysed following method development. tb was used as a model for infectious disease, with 20 tb positive and 20 tb negative samples isolated on ace chips and analysed for levels of lipoarabinomannan and ag85. using a cut-off above the negatives, the auc of roc curves were 0.9975 and 1.0, respectively. for oncology, cancer cell lines were cultured and evs isolated from supernatants were spiked into human plasma for analysis. levels of pd-l1 or glypican-1 on evs were able to be measured following ace capture. additionally, dna and rna mutations known to be present in the cell lines were able to be detected using ngs and qrt-pcr, respectively. using ad samples as a neurological disease model, tau and phospho-tau t181 (p-tau t181) in human donor plasma were detected. in 8 ad and 5 healthy donor samples, p-tau t181 signal increased 134% in diseased versus healthy donors. summary/conclusion: ace chips are an innovative ev isolation and analysis platform that allow rapid disease sample detection in a wide range of studies with high sensitivity and specificity. introduction: colorectal cancer (crc) is one of the most frequent causes of cancer-related death. in the majority of crc patients, mutation in the apc gene is among the first genetic events. it leads to uncontrolled activation of the wnt pathway, and thus, to adenoma formation. some of these adenomas may then further progress to crc with the accumulation of other mutations. the 3d organoids maintain the cellular and genetic heterogeneity of in vivo tissues and haves proved to be so far the best ex vivo model of human cancers. here we analysed the ev-based communication between cancer cells and fibroblasts by i) identifying factors that substantially increase ev release from intestinal cancer cells and ii) by determining cargo components of evs that enhance tumour cell proliferation. methods: we used commercially available and patientderived fibroblasts and crc organoids. the medical research council of hungary approved all experiments with human samples and informed consent was obtained from patients. evs were studied by using antibody-coated beads, trps, nta, tem and western-blotting. we introduced apc mutation into wild type murine small intestinal organoids by crispr-cas9. results: we found that in crc patient-derived organoid cultures, small evs were preferentially secreted. we observed that apc mutation and the accumulation of the extracellular matrix component collagen critically enhanced ev secretion in intestinal organoids. furthermore, we showed that amphiregulin, present on fibroblast-derived ev, contributed to the maintenance of the intestinal stem cell pool and to cell proliferation in epidermal growth factor-dependent crc organoids. summary/conclusion: by proving the key role of mutations, collagen deposition and ev-bound amphiregulin in the release intensity and functions of the evs, we identified novel mechanisms in the progression if crc. funding: this work was funded by otka-nn 118018, by the national competitiveness and excellence program nvkp_16-0007 (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). prostate cancer-derived evs induce a pro-inflammatory phenotype in the stroma blandine f. victor a , dolores di vizio b , andrew chin c , tatyana vagner b , javier mariscal b , mandana zandian a , catherine grasso a , roberta gottlieb a and helen goodridge a a cedars-sinai medical center, los angeles, usa; b cedars-sinai medical center, west hollywood, usa; c cedars-sinai, los angeles, usa introduction: since 90% of patients with metastatic prostate cancer (pc) develop bone metastasis, identifying the mechanism that drives this process is essential. most ev research has been focused on the role of exosomes in mediating the pre-metastatic niche formation. however, most of these studies do not separate exosomes from large evs. our preliminary studies have demonstrated that a subclass of evs known as large oncosomes (lo) can reprogram prostate fibroblasts, at the primary tumour site, promoting angiogenesis and enhancing the migration and invasion of pc cells in vitro and tumour growth in vivo. the bone marrow is the initial site of entry into the bone microenvironment for disseminating tumour cells (dtcs) and is a rich source of nutrients that houses various cells types including bone marrow derived mesenchymal stem cells (bm-msc) and immune cells such as neutrophils, which have been implicated in metastasis. here we investigate the role of lo in reprogramming bm-mscs and driving bone metastasis in pc. methods: differential centrifugation, density gradient centrifugation, trps, rna sequencing, qpcr, migration assay, invasion assay, chemotaxis assay. results: we report that pc-derived evs induce distinct gene expressions changes in bm-mscs. rna-seq analysis identified inflammatory and immune regulating cytokines as top differentially expressed genes (deg) in bm-msc. moreover, lo induced a more potent response in bm-msc in comparison to exo and to non-treated controls. the genes enriched in lo treated bm-msc were associated with tumour cell motility. in agreement with the gene expression data, lo-treated bm-msc attracted migration and invasion of significantly more pc cells than exo -treated bm-mscs. in addition, the top deg expressed in ev treated bm-msc were identified as potent neutrophil chemoattractant proteins. in line with the rnaseq findings, the lotreated bm-msc demonstrated enhanced chemotaxis of neutrophils towards them in comparison with exo or vehicle-treated bm-msc. finally, we show that the observed differences in bm-msc's response to lo and exo may be mediated by distinct molecular pathways. summary/conclusion: the results from this study provide novel insight into how tumour derived evs alter the bone marrow microenvironment and how they may drive bone metastasis in prostate cancer. the αvβ6 integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis introduction: prostate cancer (prca) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sevs). sevs isolated from prca cell media, express the epithelial-specific αvβ6 integrin, a surface receptor for fibronectin and vitronectin. the αvβ6 integrin is not detectable in healthy prostate tissues but is highly expressed in prca. in this study, we hypothesized that αvβ6 in cancer sevs plays a crucial role in angiogenesis. methods: the sevs isolated from prca cell media were characterized by nanoparticle tracking analysis, iodixanol density gradients and expression of sev markers. the αvβ6-negative endothelial cells (hmec1) were incubated with αvβ6-positive sevs from prca cells to evaluate the transfer of αvβ6 by immunoblotting (ib) and facs. the effect of αvβ6-positive sevs on motility, tube formation and angiogenic signalling were assessed by boyden chamber, angiogenesis assays and ib in hmec1. results: we demonstrate for the first time that the αvβ6 is de novo expressed on endothelial cell surface by sevmediated protein transfer. prca cell-derived αvβ6-positive sevs, significantly promote the motility and the formation of nodes, junctions and tubules by hmec1. mechanistically, we demonstrate that hmec1 treatment with sevs from pc3 cells that endogenously express αvβ6, decreases pstat1(y701), a negative regulator of angiogenesis, while upregulating survivin, an inducer of angiogenesis. hmec1 treatment with sevs isolated from pc3 cells harbouring crispr/cas9-mediated downregulation of β6, or shrna-mediated downregulation of β6, results in increased levels of pstat1(y701). this sev treatment also results in a decrease of survivin in sevs and hmec1. summary/conclusion: overall, our findings show that αvβ6 in prostate cancer sevs regulates a novel proangiogenic signalling pathway. funding: this study was supported by nci r01-224769 (lrl); p01-140043 (lrl and dca). introduction: advanced prostate cancer (pca) is asso-introduction: extracellular vesicles (evs) are secreted from cells, and carry bioactive proteins and rna cargoes. increasing numbers of studies have identified key roles for exosomes in driving aggressive tumour behaviours, including metastasis. however, the detailed mechanisms and responsible factors in the ev cargo are still unclear. recently, immune system has been considered as an important factor in establishing and maintaining metastasis. our goal is to identify the role of head and neck squamous cell carcinoma (hnscc) derived small evs (sevs) in tumour metastasis from the study analysing the effects of sevs on metastasis and tumour immunity. methods: sevs were collected from the conditioned media of hnsccs and purified through cushioned density gradient ultracentrifugation. an orthotopic mouse model was used for the assessment of tumour angiogenesis and metastasis. moc1 (inflammationinducing rarely metastasizing murine hnscc line) and moc2 (highly metastasizing murine hnscc line) were used for this study. moc1 and moc2 cells were transplanted into mice tongues orthotopically, and moc1/moc2 derived sevs or pbs were injected into the tumour twice in a week. two weeks after tumour transplantation, mice were sacrificed and tumours were sectioned for pathological analysis and facs analysis. in facs analysis, the number and species of tumour-infiltrated immune cells were measured. results: injection of sevs from moc1 into moc2 tumours suppressed frequency of lymph node metastasis. on the other hand, injection of sevs from moc2 into moc1 tumours didn't promote metastasis. cd4 positive t-cell distribution in moc2 tumour was significantly changed by moc1 sev injection. t-cell deprivation treatment using anti-cd3 antibody increased the frequency of metastasis in moc1-sev treated moc2 tumours. from the result of proteomics analysis on moc1 and moc2 sevs, immune-regulated proteins and metastasis-suppressing proteins were observed in moc1 sevs. summary/conclusion: we find that low aggressive hnscc sevs affect metastasis of highly metastasized hnscc, and also find that changing immune cell distribution may be related to the result. this mechanism and finding contributes to understanding the possible role of hnscc sevs on metastasis as well as on the tumour immune microenvironment. funding: this work was supported by the nih under award numbers r01ca163592 and r01ca206458 to aw. desmoglein 2 enhances squamous cell carcinoma tumour development through extracellular vesicles in an il-8/mir-146a-dependent mechanism introduction: the cadherin dsg2 is a stem cell marker that is upregulated in many different cancers, including sccs, and its expression correlates with poor prognosis. dsg2 activates mitogenic signalling and plays a key role in cell proliferation, migration, and survival. we recently showed that dsg2 enhances ev release, but the mechanism by which these evs modulate tumorigenesis is not fully understood. methods: we established scc cell lines stably expressing wildtype dsg2 or a palmitoylation deficient mutant, dsg2cacs. evs were isolated by sequential ultracentrifugation, iodixanol gradient separation, or qev izon column, and analysed by nta and bca. tumour xenografts were established by subcutaneous injection of 106 cells in scid mice and monitored up to 4 weeks. cytokine profiling was determined by antibody array. mirna expression was analysed by rnaseq and confirmed by qpcr. results: dsg2 enhanced ev release by 40% and promoted a~fivefold increase in tumour size in xenograft models. tumour growth was increased when control cells were treated with a single 20 µg dose of evs. loss of palmitoylation, which altered membrane trafficking of dsg2, reduced ev release (~55%) as well as tumour development. plasma evs from xenograft mice reflected in vitro particle counts from scc cell lines. a cytokine array analysis was performed revealing that dsg2-evs were enriched with pro-inflammatory cytokines including il-8, a potent chemotactic and angiogenic factor. most importantly, il-8 was surface-bound on evs. furthermore, rnaseq revealed mir-146a, a negative regulator of il-8, to be significantly downregulated in response to dsg2. treatment with mir-146a mimic or mir-146a inhibitor decreased or increased, il-8 expression in scc cells, respectively. summary/conclusion: in summary, dsg2 plays a key role in scc tumour development by increasing ev biogenesis and downregulating mir-146a, which in turn upregulates il-8 synthesis and release which can promote invasion, angiogenesis and metastasis. funding: nih r01 introduction: stem-and progenitor cell transplantation therapy holds great promise for regenerating damaged heart tissue. several lines of evidence suggest that its efficacy is mainly caused by secreted extracellular vesicles (evs). indeed, cardiac progenitor cell (cpc)-derived evs have been shown to protect the myocardium against ischaemia/reperfusion injury in several preclinical models. however, the underlying mechanisms for cpc-ev-mediated cardioprotection remain elusive. here, we utilized the proteomic composition of cpc-evs released during different culture conditions, to unravel protein-mediated effects of cpc-evs on the endothelium. methods: cpcs were stimulated with calcium ionophore (ca ion-evs) or vehicle (control-evs) for 24 hours and evs were isolated from serum-free conditioned medium using size exclusion chromatography. ev concentration and size was assessed using nta. evs were functionally characterized based on endothelial cell activation by western blotting and an endothelial cell scratch assay. the proteomic composition of both ev conditions was profiled using mass spectrometry. cpc-ev knockouts for specific proteins were generated using crispr/cas9 technology. results: we found enhanced phosphorylation of erk1/2 and akt in endothelial cells and increased wound closure after stimulation with control-evs, but not after stimulation with ca ion-evs. proteomic analysis identified a total of 1411 ev-associated proteins, with 79 proteins uniquely expressed in control-evs. another 68 proteins were revealed as candidate proteins, based on their relative enrichment in control-evs compared with ca ion-evs. go analysis demonstrated that differentially expressed proteins were involved in vascular endothelial growth factor signalling, extracellular matrix organization and angiogenesis. to investigate the involvement of the individual candidate proteins on endothelial cell activation, knockout evs of multiple proteins were generated and functionally characterized. summary/conclusion: a specific set of ev proteins is identified that may be functionally responsible for the activation of endothelial cells upon exposure to cpc-evs. generating knockout evs for each of these proteins will help to investigate their individual roles. this may lead to a better mechanistic understanding of the use of cpc-evs as therapeutics for cardiac repair. funding: erc-2016-cog-725229 evicare grant. hypoxia enhances the therapeutic potential of human cd34+ stem cell exosomes in ischaemic hindlimb repair ischaemic cardiovascular disease. we have previously shown that human cd34+ cell-derived exosomes (cd34exo) improve perfusion and function of the ischaemic tissues. hypoxia is shown to modulate the secretion and content of exosomes in both cardiovascular and cancer research. therefore, we hypothesized that hypoxia can modulate the content and regenerative efficacy of human cd34exo. methods: cd34exo were isolated from primary human cd34+ stem cells cultured under hypoxia (1.5% o2, or normoxia (20% o2, n-cd34exo) using density gradient ultracentrifugation. cd34exo size was measured using trps, nta, and dls and surface protein expression was determined using imaging flow cytometry. function of cd34exo was assessed using cell viability, migration and matrigel tube formation assays in vitro and a mouse hind limb ischaemia model (hli) in vivo. protein content of hypoxic or normoxic cd34exo was evaluated via lc-ms/ms and 2-d -dige followed by lc-ms/ms. results: we did not observe any significant differences in size or in quantity of exosomes secreted from h-or n-cd34 cells. both h-and n-cd34exo expressed cd9, cd81 and cd63 surface markers. interestingly, h-cd34exo significantly improved cell viability, migration and tube formation of huvecs in vitro compared to n-cd34exo. in the same line, h-cd34exo also significantly improved perfusion (ratio: 0.93 ± 0.05 v 0.77 ± 0.02) and prevented ischaemic limb amputation (0% v 37.5%) as compared to n-exo (p < 0.05; n = 7-8) in a murine (balbc nude) model of hind limb ischaemia. flow cytometry and confocal microscopy indicated that h-exo was uptaken by endothelial cells in the ischaemic limb. remarkably, we detected several proteins (including a fragment of hemopexin) and mirnas (mir-210) that could be responsible for the proangiogenic and beneficial function of h-cd34exo. we have also demonstrated that removal of surface proteins diminished the pro-angiogenic function of cd34exo. summary/conclusion: hypoxia enhanced the proangiogenic and regenerative potential of cd34exo, and thus, may represent a more efficient clinical strategy for cd34exo therapy. our research is clinically important to improve therapeutic angiogenesis in diabetic and cardiovascular patients with compromised stem cell populations. hyun-ji park a , jessica r. hoffman b and michael davis b a emory university, decatur, usa; b emory university, atlanta, usa introduction: exosomes, a subset of membrane nanovesicles, transfer cellular information by passing proteins and nucleic acids between cells. exosomes have been implicated as the mechanistic unit in stem cell therapy, as inhibition of exosome synthesis abrogates the effects of cell therapy following cardiac injury. more importantly, increasing evidence indicates that mirnas (mirs) within exosomes serve as important signalling molecules to regulate inflammation, recruit stem cells, and repair diseased tissue. among exosomal mirs, mir-192 and −432 are known to decrease angiogenesis, cell migration, and increase inflammation in various types of cells. here, we investigated the inhibition of these negative mirs as a means to improve the reparative capacity of c-kit+ progenitor cell (cpcs) exosomes. methods: cpcs were isolated from three paediatric patients using magnetic-bead sorting. 2ʹ-o-methylated rna duplexes inhibited mir-192 and −432 expressions in cpcs. exosomes (inhexos) were isolated from mirinhibited cpc conditioned medium. mir expression in exosomes and cpc was quantified by qrt-pcr. migration and proliferation of mesenchymal stem cells (mscs) were assessed two days post-exosome treatment. for inflammation analysis, thp1 cells with/without tnfα exposure were treated with exosomes and the expression of il-6, −8, and −10 was quantified by qrt-pcr. finally, the angiogenic potential of inhexos was tested by tube formation of cardiac endothelial cells. results: inhibitor treatment of cpcs decreased exosomal mir-192 and −432 expression. treatment with inhexos enhanced msc migration and proliferation compared with normal cpc exosome (norexo). moreover, inhexos showed promising results for immune regulation, as tnfα-induced inflammation was decreased in thp1 exposed to inhexos for 4 h. however, tube formation capacity is slightly decreased (~20%) by inhexo compared to norexo. summary/conclusion: exosomes from mir-192 and −432-depleted cpcs may be a promising strategy for the treatment of various cardiac diseases, as they enhanced stem cell recruitment and proliferation, and regulated inflammation and angiogenesis. while other studies focus on boosting the reparative potential of exosomes by increasing positive mir and mrna cargo, the inhibition of negative mir in exosomes could be an overlooked strategy for the treatment of cardiac disease. endo-lysosomes as an alternative intracellular location for ev cargo delivery with disease relevance introduction: extracellular vesicles (ev) are lipidbilayer nanovesicles that carry macromolecules and act as paracrine vectors for cell-to-cell communication. the processes regulating ev biogenesis are largely known, whereas how ev cargo is delivered to recipient cells remains poorly understood. a simple mechanism proposed is direct ev fusion with the cell membrane that liberate cargo into the cytosol. in this study, we observed that cargo release occurs also at an alternative intracellular location and that this acquires a disease relevance. methods: ev were isolated by serial centrifugation and characterized. for uptake studies, ev were traced by labelling donor cells with a lipophilic dye or by overexpressing gfp-cd63. uptake was assessed by cytofluorimetry or by live confocal imaging. co-localization studies were performed with ectopic marker expression or by immune staining. protein-protein interaction was analysed by bi-molecular fluorescence complementation (bifc). prion-like transmission was studied using a pro-fibrillogenic tau fragment in donor cells and full-length tau in recipient cells. for quantification of subcellular localization, an automated algorithm based on machine learning was developed. lysosomal stress was monitored by nuclear translocation of tfe3 and lysotracker staining. antibodies directed against pathogenic epitopes of tau were employed to assess prionlike transmission. results: ev were taken up by recipient cells through an endocytic process and accumulated in endo-lysosomes (el). when cells were exposed to ev carrying a profibrillogenic tau, recipient cells accumulated tau within el by an autophagic process. direct interaction of ev-tau and cellular tau in el favoured the appearance of pathological epitopes. cells displaying this condition showed an increased el stress and cytotoxicity. summary/conclusion: in this study, for the first time we report that el represent a critical subcellular location where transcellular prion-like transmission mediated by ev of a neurodegeneration-associated protein occurs. thus, the degradative pathway most likely involved in the recycle of ev and endogenous proteins is highjacked in disease. these findings represent a novel mechanism for ev acting as vector for transcellular propagation of tau, which opens up new therapeutic interventions trying to halt the disease. funding: supported by gelu foundation. anti-human fab fragment of cd9 antibody prevents the endocytosis of melanoma and colon cancer-derived extracellular membrane vesicles and nuclear transfer of their cargos introduction: interfering with the mechanisms regulating intercellular communication mediated by extracellular membrane vesicles (evs) may find relevance especially in oncology where cancer cell-derived evs have an implication in the malignant transformation of tumour microenvironment. our laboratories recently demonstrated a novel intracellular pathway in which a fraction of endocytosed ev-associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of rab7+ late endosomes entering into the nucleoplasmic reticulum. here, we have investigated the effect of a monovalent fab antibody against the tetraspanin cd9 (referred hereafter as cd9 fab), on the internalization of evs and nuclear transfer of their cargo proteins. chair: david r f. carter -oxford brookes university chair: neta regev-rudzi -weizmann institute of science methods: to monitor the intracellular transport of ev-associated proteins, we used bioengineered fluorescent evs containing cd9-gfp fusion protein from femx-i melanoma, sw480 colorectal cancer and bone marrow-derived mesenchymal stromal cells (msc) as donors and the same cell types as recipients. evs were enriched by differential centrifugation from 72 h serum-free conditioned media and characterized by zetaview nanoparticle tracking analysis, zetapotential and immunoblotting. cd9 fab was prepared from 5h9 hybridoma cells using the pierce fab purification kit. results: we previously demonstrated that silencing cd9 both in evs and recipient cells strongly decreased the endocytosis of evs and abolished the nuclear transfer of their cargos. here we show that cd9 fab significantly reduced the cellular uptake of cd9-gfp+ evs and the nuclear transfer of their proteins in melanoma, colorectal cancer and msc used as receptor cells in a dose-dependent manner. the effect on the nuclear transfer is probably a direct consequence of the endocytosis inhibition of evs. in contrast, the divalent, intact cd9 antibody stimulated both events. summary/conclusion: the effect of cd9 fab appears independent of the used ev-donor cell types or receptor cells, probably due to the widespread expression of cd9 both at plasma membrane and ev surface. in conclusion, by impeding intercellular communication in the tumour microenvironment, cd9 fab-mediated inhibition of ev uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti-cancer therapeutic strategies. a bright, versatile reporter for multivesicular body trafficking and exosome secretion and uptake bong hwan sung, ariana von lersner, jorje guerrero, evan krystofiak, david inmann, roxanne pelletier, andries zijlstra, suzanne ponik and alissa weaver vanderbilt university, nashville, usa introduction: live imaging of exosomes is one of the required tools to understand the function of exosomes. our previous live-cell reporter, phluorin-cd63 allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. however, there were some caveats to its use, including dim fluorescence and the inability to make cell lines that stably express the protein. methods: a stabilizing mutation, m153 r is incorporated in the phluorin moiety and now exhibits stable expression in cells and superior monitoring of exosome secretion. a dual-tag reporter was created by incorporating a further ph-insensitive red fluorescent protein, mscarlet to the c-terminus of phluo_m153 r-cd63. cancer cells stably expressing the constructs were imaged using a variety of microscopy techniques in vitro as well as in vivo. purified small evs labelled with phuo_m153 r-cd63 were imaged using immunogold transmission electron microscopy (tem) and quantitated for the half-life in the blood circulation using flow cytometry. results: phluo_m153 r-cd63 and phluo_m153 r-cd63-mscarlet are exclusively detected in exosomeenrich small ev preparations. immunogold tem visualizes the phluo_m153 r tag is located on the surface of small evs. live cell imaging reveals phluo_m153 r-cd63-positive puncta left behind migrating cells suggesting the deposition consists of exosomes. those puncta and trails are not only positive for exosome markers such as cd63, alix, and tsg101 but also correspond to small evs observed by a scanning electron microscopy. the dual-tag reporter allows visualization of the exosome lifecycle, including multivesicular body (mvb) trafficking, mvb fusion, exosome uptake and endosome acidification. summary/conclusion: using phluo_m153 r-cd63 construct, we demonstrate superior visualization of exosome secretion in multiple contexts and a role of exosomes in promoting leader-follower behaviour in collective migration by observing that exosomes are secreted at the front of migrating cells and left behind in exosome trails. the dual-tag reporter allows visualization of the entire exosome lifecycle. we anticipate that these reporters will be broadly useful to investigate regulation and functions of exosome secretion and uptake in diverse physiological conditions. funding: r01gm117916, r01ca206458, 3u19ca1795 14-05s1, r01ca216248. uncovering novel genes regulating ev-mediated functional rna transfer using a crispr/cas9-based reporter system introduction: extracellular vesicles (evs) play a pivotal role in intercellular communication through functional transfer of bioactive cargo, including rna molecules. despite increasing interest in ev-mediated rna transfer, our understanding of the pathways and mechanisms regulating ev-mediated rna delivery and processing is limited due to a lack of suitable readout systems. we recently developed a novel crispr/cas9-based reporter system that allows study of ev-mediated rna transfer at single-cell resolution. here, we further validate this system by studying the role of known targets involved in ev uptake and intracellular membrane trafficking, and subsequently employ this system to uncover various novel genes that play a regulatory role in functional rna transfer. methods: we employed a novel crispr/cas9-based stoplight reporter system, in which egfp expression is activated upon functional delivery of targeting single guide rnas (sgrnas) stably expressed by donor cells. intercellular functional rna transfer was assessed by measuring egfp expression in acceptor cells using fluorescence microscopy and flow cytometry after direct co-culture, transwell co-culture, and upon addition of isolated evs. potential roles of various genes in intercellular rna transfer were assessed by rnaimediated target knockdown in acceptor cells, prior to co-culture experiments. rnai knockdown was confirmed by qpcr analysis. results: a significant activation of egfp expression was observed in acceptor cells after direct co-culture and transwell co-culture with donor cells expressing sgrnas, as well as after addition of evs from cells expressing sgrnas. reporter activation was substantially decreased after knockdown of multiple targets involved in ev uptake through endocytosis and/or intracellular membrane trafficking. based on these results, a potential role of various novel genes in intercellular rna transfer was studied in acceptor cells. these experiments uncovered various novel targets involved in ecm binding, endocytosis, intracellular membrane trafficking, as well as various rho gtpase interactors. summary/conclusion: we previously demonstrated a crispr/cas9-based reporter system that allows the study of functional delivery of small non-coding rnas with single-cell resolution. here, we show that this novel approach allows the study of specific genetic targets and pathways in ev-mediated functional rna delivery, and unravel the regulatory pathways that dictate the underlying processes. quantitative characterization of extracellular vesicle uptake and content delivery within mammalians cells gregory lavieu a , emeline bonsergent b , eleonora grisard c and clotilde théry d introduction: extracellular vesicles (evs), including exosomes, are thought to mediate intercellular communication through the transfer of biomolecules from donor to acceptor cells. occurrence of ev-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. methods: we developed a cell-based assay in which evs containing luciferase-tagged cytosolic cargo are loaded on unlabelled acceptor cells. measurement of luciferase activity associated with acceptor cells revealed ev uptake efficacy. additional cell fractionation procedure that separates membranes from cytosol revealed the occurrence of ev-content release within the cytosol of acceptor cells. results: results from dose-responses, kinetics, and temperature-block experiments suggest that ev-uptake is limited (1% spontaneous rate at 1h), does not depend on bona-fide ev-receptor, at least for the tested acceptor hela cells. yet, further characterization of this limited ev-uptake, through cell fractionation that separates membranes from cytosol, revealed the occurrence of ev-content release within the cytosol of acceptor cells. cytosolic release is inhibited by bafilomycin-a and overexpression of ifitm proteins, which prevent virus content delivery. summary/conclusion: our results show that ev-content release requires endosomal acidification and suggest the involvement of membrane fusion. funding: anr18-ce15-0008-01 and arc pja 20171206453 and pga1 rf20180206962. introduction: glioblastoma is a highly malignant brain tumour with a poor prognosis. its ability to develop therapeutic resistance result in devastating clinical outcomes. to solve the intractable problem, we need highly sensitive diagnostics that can detect the molecular changes during treatments. extracellular vesicles (evs) can be a potential biomarker to monitor treatments and the host cell ev mapping can better reflect molecular changes in the tumour immune microenvironment. we have developed a droplet-based single ev protein sequencing platform that overcomes limitations of current bulk measurement technologies, which make it difficult to discover a rare ev population in the presence of high background. methods: we multiplex protein measurements to profile hundreds of proteins at a time by using an antibody-dna conjugate and sequencing. we barcode each ev in droplets and make amplicons that are comprised of both ev barcodes and antibody barcodes for sequencing. barcoded antibodies are made using tco-tetrazine click reaction and evs are labelled with these barcoded antibodies. the labelled evs are encapsulated into droplets with barcoded beads that serve as a template for ev barcodes. we then perform extension to make amplicons that contain both ev barcodes and antibody barcodes for sequencing. results: we successfully fabricated barcoded beads using a split-pool approach and validated by observing a fluorescence decrease of the sybr green after dna strand denaturation. we used a 3-channel droplet maker to encapsulate barcoded beads, single ev, and master mix into droplets. close packing of barcoded beads allowed >95% encapsulation into droplets. both droplet and tube-based methods achieved a similar high amplification efficiency (ct < 30 for 300 evs). we confirmed the amplicon size by running a gel, which showed the right amplicon size (~150 bp) from the droplet and tube prepared samples and no signal from the negative control. summary/conclusion: the droplet-based single ev profiling platform has the ability to identify rare immune ev subtypes in the peripheral blood, which would otherwise be impossible to detect due to the copresence of abundant normal evs. this cutting-edge technique has the potential to revolutionize treatment monitoring of high-cost immunotherapies, avoid unnecessary toxicities, and enhance personalized medicine capabilities. funding: schmidt science fellows, in partnership with the rhodes trust po1 ca069246, ro1 ca204019, r21 ca236561 quantbio graduate student award at harvard university. introduction: in this study, we compared four orthogonal technologies for sizing, counting, and phenotyping of evs. the platforms were: single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence, nanofcm nanoflow (nf), nanoparticle tracking analysis (nta) with fluorescence, and microfluidic resistive pulse sensing (mrps) . results from these platforms were compared with results from standard ev characterization techniques such as transmission electron microscopy (tem) and western blot (wb). methods: human t lymphocyte h9 (high cd81, low cd63) and promonocytic u937 (low cd81, high cd63) cells were chosen for their distinct tetraspanin profiles without abnormalities that might result from genetic manipulation. evs were isolated from culture conditioned medium (ccm) by differential ultracentrifugation (duc) and size exclusion chromatography (sec) and characterized per misev2018 guidelines. synthetic particles (silica and polystyrene spheres) with known concentrations and mixed size distributions were also tested. results: particle counts from nf and mrps were consistent, while nta detected approximately one order of magnitude lower for ccm derived evs, but not for synthetic particles. sp-iris events could not be used to estimate particle concentrations. for sizing, nf, mrps, and sp-iris returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. nta detected a population of particles with a mode diameter above 100 nm. additionally, sp-iris, nf, and mrps were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. finally, for tetraspanin phenotyping, the sp-iris platform in fluorescence mode and nf were able to detect at least two markers on the same particle. summary/conclusion: based on the results of the study, we can draw conclusions about existing singleparticle analysis capabilities that may be useful for ev biomarker development and mechanistic studies. funding: this project is funded by mh118164 and ug3ca241694. importance to ev organotropism. yet, most techniques rely on bulk characterization, or are severely restricted by the diffraction limit. the exoview r100 (nanoview biosciences) combines interferometry, immunocapture, and immunofluorescence, introduced as an alternative technique to multiplex protein detection on single evs below the limit of diffraction. here, we use this technique to characterize tetraspanin multiplexing on evs and to identify spatial patterning of tetraspanins using steric hindrance of antibodies (abs). methods: evs were isolated from conditioned media from skov-3 cell culture or human serum. evs were incubated overnight on chips to allow immunocapture by anti-cd9, anti-cd63, or anti-cd81. chips were then incubated with three fluorescent abs against the same epitopes and imaged on the exoview r100. following concentration optimization, evs were tested after preincubating with carboxy-fluorescein diacetate succinimidyl ester (cfse) or fluorescent abs against tetraspanins. results: using different concentrations of evs, binding curves could be fit to characterize binding kinetics of abs. maximum concentration of evs could be identified that minimized fluorescent overlap. bright-field interferometry (detection limit~50 nm) distinguished 10x fewer bound evs than fluorescent detection, while pre-labelling evs with cfse produced 10x more detectable evs than immunofluorescence. interestingly, evs captured by one tetraspanin did not necessarily show high fluorescent detection of the same tetraspanin. upon pre-incubating evs with a single ab, vastly different expression profiles were identified, indicating significant steric hindrance between abs. furthermore, pre-incubating evs with anti-cd9 ab significantly decreased detection of cd81 with less impact on cd63. this discrepancy indicated possible spatial patterning of tetraspanins with cd9 and cd81 closely colocalizing on the ev surface. summary/conclusion: this combination of interferometry, immunocapture, and immunofluorescence produces unique information about size distribution of evs and single ev protein profile. this data corroborates that evs have distinct subpopulations of tetraspanins and indicates that tetraspanins may be spatially patterned. regulation of liver homoeostasis, regeneration and diseases by mesenchymal stem cell-derived apoptotic extracellular vesicles university of pennsylvania, philadelphia, usa introduction: billions of cells undergo apoptosis and produce apoptotic extracellular vesicles (apopevs) each day, whereas the roles of apopevs in regulating the organismal health and disease remain poorly understood. mesenchymal stem cells (mscs) emerge as critical contributors to tissue homoeostasis, while mscs suffer from apoptosis in regenerative transplantation. in this study, we investigated the function and mechanisms of msc-derived apopevs in regulating the organismal homoeostasis. methods: fas mutant (fasmut) and caspase 3 knockout (casp3-/-) mice were applied for apoptotic and apopev deficiency. mouse bone marrow mscs were cultured and apoptosis was induced by staurosporine (sts). msc-derived apopevs were collected by serial centrifuges and were infused into mouse circulation via caudal vein. tracing of apopevs were performed by radioisotope or fluorescent labelling. liver homoeostasis was evaluated at the histological and functional aspects. liver regeneration was induced by partial hepatectomy (phx). acetaminophen (apap) was used to establish acute liver drug injury. high-fat diet (hfd) was used to establish type 2 diabetes (t2d) and non-alcoholic fatty liver disease (nafld). results: after systemic injection, msc-derived apopevs migrate to liver and can be uptaken by liver macrophages and hepatocytes. fasmut and casp3-/mice develop hepatomegaly with structural disorders, which particularly reveals hepatocyte polyploidization. furthermore, fasmut and casp3-/-mice demonstrate liver glucose and lipid metabolic disorders. importantly, msc-derived apopev infusion significantly rescues structural and metabolic dysfunction in fasmut and casp3-/-mice. mechanistically, apopevs use the soluble n-ethylmaleimide-sensitive fusion protein attachment protein receptor (snare) protein for interactions with recipient organelles thus transferring signalling molecules. moreover, msc-derived apopev infusion promotes liver regeneration after phx, prevents apap-induced liver injury, and ameliorates nafld in t2d. summary/conclusion: msc-derived apopevs serve as crucial regulators of liver homoeostasis, regeneration and diseases. these findings indicate potential significant roles of apopevs in maintaining the organismal health and in developing therapeutics for diseases. (msc-sevs) mediate osteochondral regeneration in rats. however, the therapeutic effects of these msc-sevs/exosomes in restoring the mechanical competence of the repaired cartilage for joint function in a clinically relevant animal model remain to be addressed. to investigate this, we compared the structural and mechanical properties of the repaired cartilage in a rabbit model after intraarticular administration of msc-sevs and hyaluronic acid (ha) with that of ha alone, which is widely used as visco-supplementation. methods: bilateral osteochondral defects were surgically created on 17 rabbits. immediately after surgery and at days 7 and 14 post-surgery, 9 rabbits received 1ml injections of 200 µg msc-sevs and ha in both knees, and 8 rabbits received 1-ml injections of ha in both knees. at 6 and 12 weeks, macroscopic evaluation, histological scoring and compressive testing at different points on the repaired cartilage were performed. results: defects treated with msc-sev/ha showed improvements with time in macroscopic and histological scores and mechanical properties than defects treated with ha alone. in contrast, ha treated defects showed some repair at 6 weeks, but this was not sustained, as evidenced by significant deterioration of histological scores and a plateau in mechanical properties from 6 to 12 weeks. by 12 weeks, the msc-sev/ha repaired tissues demonstrated significantly better macroscopic score (10.33 vs 7.5; p < 0.001) and histological score (21.87 vs 11.11; p < 0.001). mechanical strength as measured by the young's modulus was significantly higher in the msc-sev/ha repaired cartilage than that in ha repaired tissues [defect centre (13.41 vs 3.95mpa; p = 0.001) and overall periphery (12.22 vs 3.37mpa; p = 0.012], and approximated that of the adjacent native cartilage. summary/conclusion: our findings demonstrated that msc-sevs and ha not only improved tissue morphology of the repaired cartilage but also promoted functional mechanical competence. this study establishes a clinically translatable protocol for use of msc-sevs for cartilage repair. introduction: mesenchymal stromal/stem cell (msc)exosome (mex) treatment has shown considerable promise in experimental models of bronchopulmonary dysplasia (bpd) and pulmonary hypertension (ph). mechanisms by which mex afford their beneficial effects remain incompletely understood and here, we embark into investigating them through assessment of mex biodistribution and impact on immune cell heterogeneity. methods: newborn fvb mice were exposed to hyperoxia (hyrx, 75% o2) at birth and returned to room air at postnatal day (pn) 14. mice received a bolus mex dose at pn4. adoptive transfer studies were used to determine the role of mex-educated myeloid cells in vivo. mice were harvested at pn4, 7, 14, or 28 to characterize mex biodistribution and for assessment of pulmonary parameters. results: mex therapy effectively ameliorated core features of hyrx-induced neonatal lung injury, improving alveolar simplification, pulmonary fibrosis, vascular remodelling and blood vessel loss. exercise capacity testing and assessment of ph showed functional improvements following mex therapy. biodistribution studies demonstrated that mex localize in the lung, where they interact with lung monocytes/macrophages. whole lung mass cytometry (cytof) revealed that mex treatment promotes a pro-homoeostatic shift in lung immune cell apportion, replenishing the early hyrx-induced depletion in pulmonary cd45+ immune cells, restoring alveolar monocyte and macrophage populations and suppressing cellular inflammation. ex vivo and in vivo analysis showed that mex promotes a "pro-resolving" ccr2-monocyte phenotype. notably, adoptive transfer of mex-educated bone marrow-derived myeloid cells (bmdmy), but not naïve bmdmy, restored alveolar architecture, blunted fibrosis, improved vascular remodelling and pulmonary blood vessel loss. summary/conclusion: mex treatment ameliorates core features of experimental bpd, restoring lung architecture, decreasing pulmonary fibrosis and vascular muscularization, ameliorating ph and improving exercise capacity. the beneficial actions of mex are associated with modulation of immune cell phenotypes, arising from mex-monocyte interaction. furthermore, adoptive transfer of mex-educated bmdmy rescued, at least in part, alveolar architecture, reduce fibrosis, improve vascular remodelling and pulmonary blood vessel loss. funding: this work was supported in part by an american thoracic society foundation grant (grw); the little giraffe foundation (grw); charles h. hood foundation major grants initiative (sk), nih r01hl146128 (sk) and united therapeutics research grant (sk and sam). immunomodulatory small extracellular vesicles derived from mesenchymal stem cells: a potential cell-free therapy for acute and chronic pulmonary vascular diseases introduction: vascular inflammation plays a critical role in acute respiratory distress syndrome (ards) and pulmonary arterial hypertension (pah). despite decades of research, there is no curative therapy for either condition. mesenchymal stem cells (mscs) have shown preclinical efficacy, mediated by release of extracellular vesicles. hence, msc-derived small extracellular vesicles (sevs) can harness the benefits of mscs with advantages in cost and safety. this study aims to evaluate the immunomodulatory effects of sevs in preclinical ards and pah. methods: msc-sevs were characterized by nanoparticle tracking analysis, electron microscopy and western blot. live fluorescence imaging measured in vitro and in vivo distribution of sevs. using a lipopolysaccharide (lps)-induced mouse model of acute lung injury (ali), a time course study of inflammatory response guided endpoint analyses. cell count and cytokines were measured in bronchoalveolar lavage fluid (balf) and histological lung injury was assessed. in ali mice, saline, mscs, msc conditioned media or sevs were administered 0.5 h post-lps. using a monocrotaline (mct)-induced rat model of pah, animals received saline or sevs at day 3. haemodynamic changes and right ventricular hypertrophy were evaluated at 3 weeks. results: msc-sevs were 72 nm in size with cd63/81 expression. pkh26-labelled sevs were taken up by endothelial cells. in the ali time course study, cell count and il1b in balf peaked at 24h post-lps, whereas il6 peaked at 10h. histology showed significant intra-alveolar cell infiltrate at 72h. msc conditioned media attenuated il1b in balf, whereas a trend towards reductions in il1b and cell count were seen from delivery of mscs and sevs. using fluorescence imaging, lung accumulation of dir-labelled sevs was highest when administered 1 h post-lps as compared to 5 h, 10 h or 24h. for pah rats, sevs reduced right ventricular systolic pressure (51.4 ± 8.3 mmhg) as compared to control (68.1 ± 0.4 mmhg; p = 0.012), whereas no changes were observed for right ventricular remodelling. summary/conclusion: these findings demonstrate the potential of msc-sevs to be used as a cell-free immunomodulatory therapy for acute and chronic lung vascular diseases. additional live and ex-vivo biodistribution studies will determine optimal timing of sev administration, tissue distribution and clearance in both ali and pah. changes in extracellular vesicle protein cargo after pro-inflammatory priming of umbilical cord mesenchymal stem cells (ucmscs) have been shown to suppress inflammatory responses in studies of autoimmune diseases. these therapeutic effects can be attributed to paracrine signalling, by which extracellular vesicles (evs) are one of the essential components. this study looks at how the culture conditions of ucmscs affects the type of evs they secrete. it also aims to identify an ev population with an anti-inflammatory potential for the treatment of autoimmune diseases. methods: ucmscs were isolated and culture expanded in a quantum® cell expansion system, then grown at 21%o2, 5%o2 and primed with a pro-inflammatory cocktail. evs were isolated from ucmsc conditioned media by differential ultracentrifugation using a sucrose cushion and characterised by transmission electron microscopy and nanoparticle tracking analysis. ev markers were analysed using a europium-based immunoassay, macsplex exosome detection kit and immunoblotting. a proximity-based extension assay was used to identify 92 inflammatory proteins in the evs. results: there was no difference in evs cultured at 21%o2, 5%o2 or with pro-inflammatory conditions when analysed for size and morphology. all evs displayed the tetraspanin markers (cd9/63/81) and internal proteins (alix, hsp70). evs from primed cells showed a > twofold increase of 5 cc chemokines and a > sixfold increase in cxcl5 and csf-1. protein cargo did not differ in evs from 21%o2 and 5%o2. summary/conclusion: this study showed that proinflammatory culture conditions alter ev protein cargo, evidenced by the increased production of chemotactic and angiogenesis associated proteins. upcoming rnaseq analysis will show if ucmsc culture conditions also affect mirna expression in evs. ongoing functional studies will determine how changes in ev cargo correlates with changes in t-cell proliferation and polarisation. funding: this work is fund by the orthopaedic institute ltd, keele university and the rjah orthopaedic hospital charity. alzheimer's disease biomarkers in plasma extracellular vesicles of neuronal origin correlate with brain pathology in mice introduction: multiple studies have shown that neuronal-derived extracellular vesicles (ndes) in blood contain alzheimer's disease (ad) biomarkers, especially tau. however, the convergent validity of tau in blood ndes in relation to brain pathology is yet to be determined. to address this, we measured total and phosphorylated tau levels in matched nde and brain tissue samples from ad mouse models. methods: we collected the cortex, hippocampus and plasma of 4 2xtg-ad, 15 3xtg-ad, and 9 wild type (total of 28 mice; 14 female, 14 male; age: mean = 8.17, sd = 1.61, 5-11 months) . plasma samples were collected retro-orbitally for 5 weeks and at euthanasia via heart puncture. ndes from the pooled serial blood collections (nde1) and the single endpoint (nde2) were immunocaptured by targeting the neuronal marker l1cam. we measured human total tau and pthr181-tau (p-tau) in ndes and cortex and hippocampus homogenates using a luminex multiarray. results: overall, there were strong positive correlations for both total tau and p-tau between ndes and brain tissues across mice types. total tau in ndes showed positive correlations with levels in the cortex and hippocampus (r = 0.6885 and 0.7026, p < 0.0001, cortex vs nde1 and nde2; r = 0.4958, p = 0.0073, hippocampus vs nde1; r = 0.6058, p = 0.0006, hippocampus vs nde2). levels of p-tau in nde1 showed positive correlations with levels in the cortex (r = 0.4640, p = 0.0155) and hippocampus (r = 0.6086, p = 0.0008); however correlations were not observed for nde2 (r = 0.1152, p = 0.6273 vs cortex; r = 0.0531, p = 0.7926 vs hippocampus). summary/conclusion: tau levels in circulating ndes reflect levels in cortex and hippocampus across ad model mice, supporting their convergent validity as "liquid biopsy" biomarkers for ad. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. exosomal ceramide mediates neurotoxicity of amyloid beta (aβ) in alzheimer's disease. ahmed elsherbini a , simone crivelli b , alexander kirov c , michael dinkins d , zhihui zhu a , haiyan qin a , sanjib karki a , priyanka tripathi a and erhard bieberich a a university of kentucky, lexington, usa; b university of kentucky, lexington, usa; c augusta university, augusta, usa; d augusta university, augusta, usa introduction: amyloid beta is a pathologic hallmark of alzheimer's disease (ad), however, the mechanism of aβ neurotoxicity is not fully understood. it has been reported that exosomes associate with aβ, but it is not clear how this association affects aβ neurotoxicity. methods: here we utilized several techniques to isolate exosomes from the sera of wild type (wt) and ad transgenic mouse model. (5xfad) as well as alzheimer's patients and healthy controls. we used exoquick, exoeasy, sequential ultracentrifugation, and size exclusion chromatography. particles' size and number were characterized by nanoparticle tracking analysis (zetaview). results: we report that the sphingolipid ceramide mediates neurotoxicity of aβ. we show that sera from ad transgenic mouse model (5xfad) and ad patients, but not the wt or healthy controls, contain a subpopulation of astrocyte-derived exosomes that are enriched with ceramide and are prone to aggregation (termed astrosomes) as confirmed by nanoparticle tracking and cluster analyses. when taken up by introduction: multiple sclerosis is the most common chronic inflammatory demyelinating disease of the central nervous system, affecting more than 2 million people worldwide. ms is a multifactorial, immunemediated disease caused by complex genetic and environmental interactions. in recent years, extracellular vesicles (evs) have been described as powerful mediators of the modulation of biological processes (e.g. inflammatory and immune response) following environmental exposures such as particulate matter (pm), and have been described altered in ms. we characterized evs in 48 patients with ms and 20 healthy subjects matched for age and gender and evaluated the effects of pm exposure on ev release patients with ms compared with controls. methods: evs isolated from blood samples were characterized by nanotracking analysis and by flow cytometry after labelling with the following markers: cd14 + (monocyte), cd61+ (platelet), cd66+ (neutrophil), cd25+ (t-reg), and cd105+ (endothelium). pm10 and pm2.5 concentrations at the residency of each subject were obtained from the regional air quality monitoring network. results: we observed decreased concentrations of cd14+ (p < 0.05), cd61+ (p < 0.003), cd66+ (p < 0.009), cd25+ (p < 0.003), and cd105+ (p < 0.003) in patients compared with controls. in cases, pm was inversely associated with cd14+ evs (pm2.5, β = −0.03; p < 0.05), cd66+ evs (pm2.5 β = −0.03; p < 0.03), and cd25+ evs (pm10 β = −0.02; p < 0.04; pm2.5, β = −0.03; p < 0.02). on the contrary, in controls pm was positively associated with cd14+ evs (pm10 β = 0.02; p < 0.03; pm2.5, β = 0.03; p < 0.02). summary/conclusion: our findings showed a different composition of blood-derived ev subpopulations in patients compared with controls. moreover, we observed that patients and controls react differently to pm exposure in terms of blood-derived ev release, suggesting the involvement of this mechanism in the modulation of both inflammatory and immune responses, and thus in ms pathogenesis. plasma neuronal and astrocyte-derived exosomes serve as biomarkers of neurodegeneration and systemic bioenergetic effects in male cynomolgus monkeys self-administrating oxycodone ashish kumar a , yixin su a , david soto-pantoja a , jingyun lee a , ravi singh a , cristina furdui a , michael nader b and gagan deep a a wake forest baptist medical center, winston-salem, usa; b wake forest baptist medical center, winston-salem, usa introduction: opioid use disorder (oud) is currently a health emergency in the usa affecting millions of people. oud is a complex issue requiring a multipronged strategy. at the biological level, there is an urgent need to understand the dynamic molecular changes and adverse effects associated with opioid addiction. here, we aimed at identifying the biosignature of brain cells-derived exosomes associated with opioid addiction in a non-human primate (nhp) model of oud in which cynomolgus monkeys perform cognitive tasks and self-administer (sa) intravenous oxycodone daily. we also characterized the systemic adverse effects of the brain cells-derived exosomes from drug-naïve and oxycodone sa monkeys. methods: we isolated total exosomes (te) by ultracentrifugation and exoquick methods from the plasma of male monkeys self-administrating oxycodone for 3 years and naive monkeys. subsequently, from the te population, we isolated neuron-derived exosomes (nde) and astrocytes-derived exosomes (ade) using surface biomarkers l1cam (l1 cell adhesion molecule) and glast (glutamate aspartate transporter), respectively. this novel method involved streptavidin coated magnetic beads and photo-cleavable (pc) biotin, providing us biologically intact exosomes useful for co-culture studies. we characterized the exosomes by nanoparticle tracking analyses (nta), western blotting, flow cytometry, immunogold labelling, transmission electron microscopy (tem), elisas and mass spectrometry. respirometric profiling in cardiac myoblasts and monocytes following exosomes treatment was performed by seahorse xf. results: the quality of isolated exosomes (te, nde, and ade) was confirmed by nta (size distribution and concentration), western blotting (e.g. cd9) and tem (size and shape). nta did not show any significant difference in exosomes size and concentration (number per ml) between control and oxycodone sa groups. flow cytometry (e.g. l1cam and glast) and immunogold labelling (cd9, cd63 and l1cam) confirmed the purity of nde and ade isolated from te. proteomics analyses of te, nde and ade identified several unique proteins present in exosomes from the oxycodone sa group. interestingly, we observed significantly higher expression of neurodegeneration markers neurofilament light protein (nfl) and alpha-synuclein in nde and ade of oxycodone sa group compared to controls. furthermore, te treatment of h9c2 cardiac myoblasts and raw264.7 monocytes significantly compromised their mitochondrial metabolism (basal and maximum respiratory capacity). summary/conclusion: these results suggest the utility of plasma exosomes as biomarkers for better understanding of the neurodegenerative and systemic effects of oxycodone addiction. funding: da049267, da017763. vesicles released during mycobacterium tuberculosis infection: immunomodulatory (glyco)lipids and role in host-pathogen interactions emilie layre, pierre boyer and jerome nigou cnrs-université paul sabatier, toulouse, france introduction: the tuberculosis disease remains one of the top 10 causes of death worldwide. mycobacterium tuberculosis (mtb) has evolved strategies to evade immune responses and to persist within the hostile intracellular environment of alveolar macrophages. the current lack of efficient anti-tuberculosis strategies is largely due to our incomplete understanding of the host-pathogen interactions of mtb infection. vesicles released by the bacillus itself (bacterial membrane vesicles, bmv) and by infected cells (host extracellular vesicles, hev) have immunomodulatory properties in vitro and when administered to animals. if vesicles likely play key role in host-pathogen interactions of the tuberculosis infection, their content in bacterial factors, their uptake, trafficking and interaction with host cells receptors remain incompletely deciphered. methods: bmv and hev have been purified by combining differential centrifugation, density gradient and exclusion chromatography. after characterization by microscopy, nanosight and western blot, their content in bacterial (glyco)lipids has been characterized by the use of high sensitivity mass spectrometry-based lipidomic approach. bmv have been tested for their capacity to activate reporter cell lines of pattern recognition receptors. in addition, fluorescent-labelled bmv have been used to study their uptake by host cells thank to super-resolution microscopy. results: we have undertaken to characterize the content, the trafficking and interaction with pattern recognition receptors of bmv and hev released during infection by mycobacteria of variable virulence. we have importantly optimized the purification of bmv showing that lipoproteins aggregates are co-purified with vesicles on density gradient. sfc-ms lipidomic analyses allowed the characterization of the repertoire of immunomodulatory bacterial lipids released by bmv and hev, which excluded a continuum between these two release pathways. preliminary, assays have shown that these vesicles are capable to interact with different pattern recognition receptors including tlr and lectins. finally, we have been able to visualize fluorescent-labelled vesicles uptake by macrophages using superresolution microscopy. summary/conclusion: during m. tuberculosis infection, the bacillus as well as infected cells release vesicles that harbour different content in immunomodulatory bacterial (glyco)lipids, including strain-specific lipids. these vesicles likely play important role in host-pathogen interactions by modulating immune response beyond the infected cells, in part through their interaction with different pattern recognition receptors. funding: fondation pour la recherche medicale, fondation fonroga. introduction: conventional diagnoses of mycobacterium tuberculosis (mtb) rely on quantifying bacteria in sputum samples, which make it incapable of measuring the body's total bacterial load and diagnosing patients that have difficulty producing sputumsuch as children and those that are hiv-positive. nanoscale (30-200 nm) outer membrane vesicles (omvs), which are shed from their bacterial cells of origin and circulate in the bloodstream, have been found to contain rich molecular information from their mother cells. despite the diagnostic potential, their nanoscale size in the presence of high background has complicated the use of these promising biomarkers for clinical diagnosis of tuberculosis. chair: amy buck -the university of edinburgh chair: cherie blenkiron -the university of auckland methods: here we report two complementary approaches to systematically discover and clinically detect mtb-derived omvs using protein and rna biomarkers. first, we employ a digital droplet elisa on whole, unprocessed samples to detect and quantify the presence of these omvs using surface protein markers. second, we have developed a platform to specifically enrich for mtb-derived omvs using our previously developed magnetic nanopore platform, wherein millions of nanofluidic devices are operated in parallel, increasing throughput relative to a single nanofluidic device by a million-fold. using this approach, we identify rnas that are specifically enriched in mtb-derived omvs and can be used to identify tb strain, infectious activity, and total body burden. results: using these platforms, we enriched for mtbderived omvs from plasma and profiled their cargo, both proteins and rna. we first determined a panel of protein biomarkers for multiplexed detection of omvs through a digital droplet sandwich elisa. we then tested our protein markers on spiked plasma samples as models for clinical tb samples. simultaneously, we performed rna sequencing and discovered a panel of rna biomarkers that are preferentially enriched in omvs. we picked ten of the most highly-expressed rna biomarkers and also tested for them on spiked plasma samples using our magnetic nanopore platform. summary/conclusion: these results demonstrate the capability of omv biomarkers in the development of novel liquid biopsy based mtb diagnostics. building on this work, we are working with clinical collaborators to test our assays on clinical samples from philadelphia and west africa. funding: nih ot08.3 introduction: a dearth of knowledge exists regarding the molecular mechanisms by which host exosomes regulate immune response to infections caused by gram-negative pathogens. to address this gap in knowledge, our laboratory has been using two wellestablished model organisms; yersinia pestis (yp), and burkholderia pseudomallei (bp). yp and bp cause the emerging human diseases plague and melioidosis respectively. currently, no licenced vaccines or highly effective therapeutics are available for either disease. methods: ex were purified from naïve u937 monocytes (exu) and infected u937 (exi) by serial centrifugation followed by sucrose density gradient purification, and characterized by tem, zetaview nanoparticle tracking, and exosome markers (cd63, tsg101, . immune responses of naïve u937 cells and response mechanisms were analysed following treatment with equivalent amounts of exi or exu (as control). these included macrophage differentiation assays, multiplex measurements of inflammatory cytokines, bacterial clearance assays, quantitative protein microarray analysis of 173 host signalling proteins, sirna knockdown of exi-induced cytokines in recipient cells, and mass spectrometry (ms) analysis of exi contents. for all assays, at least four biological replicates were performed. results: exi induce monocyte differentiation to macrophages and dramatic release of il-6, il-8 and il-10 cytokines, effects that are also seen when monocytes are infected with the bacteria. the exi also induce a substantial increase in the capacity of the recipient monocytes to clear bacteria in an il-6-dependent manner. specific host signalling molecules are strongly modulated by the exi, including p38, jak2 and alk for exi-yp, influencing the observed phenotypes. ms analysis showed lack of lps in exi-yp and demonstrated the presence of specific bacterial proteins that have antigenic properties. summary/conclusion: we have identified some of the molecular mechanisms by which exi assist the host in clearing infection. exi prime distant naïve monocytes through modulation of distinct pathways such as p38 to mount immune responses similar to when they become infected. these include differentiation to macrophages and migration to infection site for increased il-6-dependent bacterial clearance. introduction: recruitment of monocytes to sites of infection is important in restricting growth and invasion of various microorganisms such as pathogenic fungi c. albicans. beside complement supported phagocytosis and extracellular trap formation, human monocytes secrete extracellular vesicles which are crucial in cellular communication in physiology and pathophysiology as they transfer proteins, lipid, and nucleic acids. the current study attempts to shed light on immune evasion mechanisms by c. albicans via extracellular vesicles. methods: human monocytes were isolated by magnetic beads technique and extracellular vesicles were isolated using polymer precipitation or ultracentrifugation or size exclusion chromatography. vesicles were characterized by elisa, lc/ms-based proteomics, confocal laser scanning microscopy (clsm) as well as electron-and dynamic light scattering microscopy, etc. crispr-cas9 based genome editing was performed to knockout cd11b in human monocytic thp-1 cells. effect of isolated vesicles were determined by using proximity ligation assay (pla), elisa, western blot, next generation rna sequencing, qpcr, immunohistochemistry, etc. results: here we show for the first time that human blood derived monocytes alone and in a whole blood model strongly induced and released extracellular vesicles in response to the pathogenic fungus c. albicans. one induced population carried the anti-inflammatory cytokine tgfβ-1. release of these vesicles is triggered by binding of soluble β-glucan from c. albicans to the cr3 receptor on monocytes as demonstrated by crispr-cas9 based cr3 genome editing in thp-1 cells, and by using cr3 knock out mice. isolated tgf-β1-transporting vesicles reduced the inflammatory response in human m1 macrophages and in a whole blood model. the anti-inflammatory effect by tgf-β1transporting vesicles is investigated in detail and results in inhibition of il-1β gene transcription. summary/conclusion: showing that human apoptotic bodies similarly induced tgf-β1-transporting vesicles from human monocytes we hypothesize that c. albicans hijacks this new cr3-dependent anti-inflammatory vesicle pathway for immune escape. funding: this work was supported by the "deutsche forschungsgemeinschaft" transregio funginet 124 projects c4, c5, c6 and z2. introduction: to date, most research involving extracellular rnas has focused in rnas encapsulated inside extracellular vesicles (evs) or in total unfractionated biofluids. it is known that exrnas also exist outside vesicles or in lipoprotein particles. however, nonvesicular exrnas remain widely uncharacterized despite being a feasible source of contaminants in ev preparations. our interest in nonvesicular exrnas arises from the observation that some small rnas, such as specific trna-derived fragments, have much higher relative representation in this extracellular fraction. at least in part, this enrichment seems to be a consequence of their differential extracellular stability. methods: to get a representative picture of the whole set of rnas released to the extracellular nonvesicular space by cultured human cells, we inhibited extracellular degradation by adding recombinant ribonuclease inhibitor to the cell-conditioned media and studied the kinetics of rna release and degradation. high-resolution iodixanol gradients were used to separate evs from extracellular rnps or vesicle-free rna. the conversion rate between parental ncrnas and their fragments was studied by high-throughput sequencing and northern blot. results: the inhibition of extracellular rnase activity revealed the presence of full-length trnas and ribosomes in the extracellular space of a variety of malignant and non-malignant cell lines. extracellular ribosomes co-isolate with evs purified by ultracentrifugation or size-exclusion chromatography, but not with evs purified by density gradients.these ncrnas are substrates of extracellular rnases, demonstrating an extracellular biogenesis route for the formation of ncrna-derived fragments, some of which achieve remarkable stability and can be detected in biofluids. we also highlight the immunoregulatory potential of purified rna-containing extracellular complexes. summary/conclusion: in conclusion, ribonuclease inhibition dramatically shapes extracellular rna profiles and uncovers a population of extracellular ribosomes, trnas and other coding and noncoding rnas which exists outside evs. although these rnas are prone to degradation, some of their fragments can accumulate in cell culture media and in biofluids. this dynamic view of exrnas impacts our understanding of rna secretion mechanisms and may offer a window to new molecules with biomarker potential. in contrast, evs confer an rnase-protected environment and contain more full-length ncrnas (trnas, yrnas, 7sl rnas, rrnas depending on vesicle size) than their fragments. introduction: cd47 is a ubiquitously expressed membrane protein that functions as a receptor for thrombospondin-1 and the counter receptor for signal regulatory protein-α in phagocytes. high expression of cd47 is associated with a poor prognosis for some cancers. conversely, cd47 blocking agents are in clinical trials for enhancing innate and adaptive antitumor immunity in cancer patients. these studies suggest utility of cd47 as a diagnostic and prognostic biomarker and as a therapeutic target. cd47 is also expressed on extracellular vesicles (evs), and we reported that cd47 expression identifies a distinct population of evs from those that express the traditional ev markers cd63 or mhc1. cd47-, cd63-and mhc1-enriched vesicles contain distinct small rna populations (pmid: 29416092), and these differ in rna content from evs that lack any of these markers. the mechanisms by which cd47 directly or indirectly regulates which rnas are packaged into ev remain unknown. methods: to elucidate the mechanism by which cd47 regulates ev rna composition and function, we performed global mirna microarray analysis between evs produced by wild type and cd47-deficient t cells. results were further validated using real-time pcr and rna-immunoprecipitation. interactions between cd47 and exportin-1/ran complex was identified by mass spectrometry and confirmed by using co-immunoprecipitation, subcellular localization, flow cytometry, and confocal and electron microscopy. results: ev released from cd47-deficient human t cells and in cd47-/-mouse plasma were enriched in 5ʹ-7-methylguanosine-capped micrornas and mrnas that depend on the exportin-1/rangtp pathway. knockdown of cd47 in wild type cells or thrombospondin-1 treatment correspondingly enhanced levels of capped-rnas released in ev and re-expressing cd47 in null cells decreased their levels. mass spectrometry and co-immunoprecipitation identified specific interactions of cd47 with components of the exportin-1/ ran nuclear export complex and its known cargos and between the cd47 cytoplasmic adapter ubiquilin-1 and the exportin-1/ran complex. interaction of cd47 with exportin-1 was inhibited by leptomycin b, which inactivates exportin-1 and increased levels of cap-dependent rnas in ev released from wild type but not cd47-deficient cells. summary/conclusion: these findings indicate that cd47-dependent thrombospondin-1 signalling regulates cytoplasmic levels of cap-dependent rnas in t cells at least in part through ubiquilin-1-and gtpdependent physical interactions with the exportin-1/ ran transport complex, which regulate levels of specific pre-mirnas and mrnas available for sorting into evs. funding: this work was supported by the intramural research program of the nih/national cancer institute (zia sc 009172). role of membrane protein palmitoylation in extracellular vesicle biogenesis in squamous cell carcinoma introduction: desmoglein 2 (dsg2), is a palmitoylated cadherin that is involved in cell-cell adhesion. interestingly, dsg2 promotes mitogenic cell signalling and is upregulated in many cancers, including scc, contributing to poor prognosis and survivability. we recently demonstrated that dsg2 promotes ev release, but the mechanism by which dsg2 enhances ev biogenesis and role of palmitoylation is poorly understood. methods: pharmacological drug inhibitors 2-bromopalmitate, gw4869, and bafilomycin a1 were used. stable scc cell lines were established by retrovirus infection expressing gfp, wild type dsg2/gfp, or palmitoylation deficient dsg2cacs/gfp. evs were isolated by sequential ultracentrifugation and iodixanol gradient separation and analysed by nta. proteins associated with the endocytic pathway were analysed by immunofluorescence and imaged by confocal microscopy or immunoblotting and signals were quantitated using imagestudio. results: here we demonstrate that the effect of dsg2 on ev release was reduced by the palmitoylation inhibitor 2-bromopalmitate. furthermore, mutations that prevented palmitoylation (dsg2cacs) dramatically abrogated ev release by targeting of un-palmitoylated dsg2 to the lysosomes for degradation. dsg2 increased expression and subcellular localization of flot1, a membrane lipid raft protein critical for membrane invagination. dsg2 also altered membrane localization of several early (eps15 and eea1), but not late (rab7, rab11, and hrs), endocytic pathway proteins. loss of palmitoylation in the dsg2cacs mutants abrogated these effects. finally, dsg2-induced ev release was abrogated by the sphingomyelinase inhibitor gw4869 or augmented by the v-atpase inhibitor bafilomycin a1. summary/conclusion: the combined results of the drug treatments and functional mutations of dsg2 suggest that dsg2 plays a critical role in ev biogenesis by modulating proteins involved in early endosome sorting and is dependent on post-translational palmitoylation. introduction: the translation initiation factor eif4e (4e) is an oncogenic protein that is upregulated in 30% of cancers including a subgroup of acute myeloid leukaemia (aml) patients. eif4e regulates post-transcriptional rna processing including the nuclear export and/or translation of mrna transcripts. in particular, it selectively increases the expression of genes that have a prominent role in cancer progression such as myc, cyclin d1, and mcl1. furthermore, our lab pioneered studies demonstrating that a subset of 4ehigh aml patients is clinically responsive to treatment with a 4e inhibitor (ribavirin) indicating the importance of 4e in aml progression and its relevance as a therapeutic target. we investigated an as yet unexplored perspective of 4e-whether the oncogenic role of 4e is in part mediated by its function as a master regulator of vesiculation. methods: to assess mrna export and identify 4ebound mrna targets that correspond to vesiculationrelated genes and associated cargo, we used cellular fractionation and rna immunoprecipitation techniques. to determine whether 4e regulates the number of extracellular vesicles (evs) released as well as their protein and rna cargo we used nanoparticle tracking analysis (nta) as well as mass spectrometry, antibody microarrays, and rna sequencing technologies. results: eif4e upregulates cellular protein levels of the vesiculation marker cd63 by increasing its nuclear export. in addition to increased cellular expression, cd63, cd81, cd9, and flotillin-1 proteins are elevated in evs released from 4e-high cells. this is also associated with an increased release of vesicles that are 50-200 nm in size. currently, we have validated the upregulation of several receptors and cytosolic proteins in evs isolated from 4e-overexpressing cells that function in cell growth, migration, invasion, and stemness. the most abundant rnas in our ev preparations are micrornas (mirs) and we have confirmed downregulation of several of these. summary/conclusion: our work shows that 4e reprograms the vesiculation of cancer cells changing the release and cargo of evs. this may impact cellular communication and tumour biology, which we are currently addressing in functional studies. we hope that these studies will highlight novel therapeutic strategies for aml patients. intranasal administration of neural stem cells-derived extracellular vesicles promotes neurogenesis and reduces neuroinflammation and amyloid plaques in a mouse model of alzheimer's disease introduction: cognitive and memory impairments worsen with time in alzheimer's disease (ad), likely due to a progressive loss of hippocampal neurogenesis, and escalation of neuroinflammation. these changes are also accompanied by increased deposition of amyloid plaques in the brain. methods: in this study, using the 5xfad mouse model, we examined the efficacy of extracellular vesicles (evs) shed from the rat subventricular zone neural stem cells (svz-nscs) for disease modification. we first purified evs from the rat svz-nsc cultures through ion-exchange chromatography and then administered intranasally to 3-months old 5xfad mice (~75 billion/week for two weeks). two months later, the functional effects of ev treatment were quantified through a series of behavioural tests, and animals were euthanized for quantification of hippocampal neurogenesis, oxidative stress, neuroinflammation, and amyloid plaque deposition. results: in comparison to ad mice receiving vehicle, ad mice receiving nsc-evs displayed improved cognitive function to discern minor changes in the environment in an object location test, better spatial recognition memory in an object-in-place test, and improved pattern separation ability in a pattern separation test. besides, ev-treated ad mice displayed no anhedonia in a sucrose preference test. analyses of neurogenesis using the birth-dating marker 5ʹ-bromodeoxyuridine and the newly born neuron marker doublecortin revealed maintenance of a higher level of hippocampal neurogenesis in ad mice receiving evs, in comparison to vehicle-treated ad mice. moreover, analyses of brain tissues from ev-treated ad mice revealed decreased concentrations of oxidative stress markers malondialdehyde and protein carbonyls and elevated levels of antioxidants catalase and superoxide dismutase. also, the concentration of proinflammatory cytokines tumour necrosis factor-alpha and interleukin-1 beta and the extent of amyloid plaques were significantly reduced in ev treated ad mice. immunohistochemical analysis showed reduced hypertrophy of astrocytes. summary/conclusion: intranasal administration of nsc-derived evs restrains the deterioration of cognitive and mood dysfunction of ad by maintaining higher levels of neurogenesis and curtailing the progression of neuroinflammation. funding: supported by a grant from the national institute of neurological disorders and stroke (1r01ns106907-01 to a.k.s.) ot10.2 the case of mesenchymal stromal cells, opening new perspectives in the use of ipsc in regenerative medicine. the aim of this study is to evaluate the potential of ipsc-ev in the treatment of kidney disease. methods: the ipsc were generated from skin fibroblast after informed consent of healthy donors (cytotune®-ips 2.0 sendai reprogramming kit -protocol: clementino fraga filho uh 38583914.7.0000.5257/933.018). the ev were isolated from ipsc supernatants (cultured 24 h in mtesr-1 medium) by ultracentrifugation (100,000 g for 2 h at 4°c). characterization of ipsc-ev was performed using zetaview, tem, exoview™ tetraspanin kit and macsplex exosome kit. for in vitro injury, renal epithelial cells were cultured under hypoxia (1% o2). for in vivo injury, male wistar rats were submitted to bilateral renal arterial clamping (45 min) followed by reperfusion without or with injection of ipsc-ev (protocol approval: federal university of rio de janeiro -043/19). kidney damage was assessed by histological and immunohistochemistry analyses (pcna, tunel and ed-1). modulation of rna levels was assessed by rt2 profiler pcr array. results: the results show that ipsc-ev reduce renal cell death, tissue damage, macrophage infiltration, promote mitochondria protection and ameliorate renal function. the ipsc-ev mechanism of action is related to the regulation of key genes known to prevent damage caused by oxidative stress like gstk1, sod1, sod3, txn1 and txnrd2. characterization of ipsc-ev showed that ipsc-ev can carry important molecules that can support renal recovery as epcam and prominin-1. summary/conclusion: ipsc-ev presents renoprotective properties, acting on different aspects of aki. this presents a new relevant application of ipsc as a source of ev for therapeutic purpose in kidney diseases. the hospital for sick children, toronto, canada introduction: incomplete lung development, also known as pulmonary hypoplasia (ph), is a recognized cause of neonatal death. we have previously shown that experimental ph can be rescued by the administration of extracellular vesicles derived from amniotic fluid stem cells (afsc-evs) through an rna-mediated mechanism. this effect was not observed with evs derived from mesenchymal stromal cells (msc-evs) . the aim of this study was to 1) evaluate which rna species were responsible for ph rescue, and 2) to define the mechanism behind this effect. methods: evs were isolated and characterized from conditioned medium of rat afscs and rat mscs (control group) using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd63, hsp70, flo-1, and tsg101 (western). to identify the mediators of afsc-evs, we used deseq (fdr<0.01) to differentially analyse rna from: a) asfc-ev and msc-ev cargo, isolated with seramir and sequenced with nextseq. b) lung epithelial cells from rat ph lungs treated with vehicle or afsc-evs. epithelial cell rna was isolated with mirvana and sequenced with nextseq. we correlated afsc-ev cargo mirna with validated mrna targets that were downregulated after ev conditioning in lung epithelial cells. results: of the rna species contained in asfc-ev and msc-ev cargo, mirnas were the most proportionally different between the two ev populations. afsc-evs were enriched for mirnas that are critical for lung development, such as mir17~92 and their paralogues that control lung branching morphogenesis. afsc-ev administration to ph lung cells significantly downregulated 35 genes, which formed 415 mirna-mrna reported interactions. summary/conclusion: afsc-evs contain many rna species in their cargo, but mirnas are the main effectors of their ability to rescue underdeveloped foetal lungs. we have identified for the first time that afsc-ev biological effect on underdeveloped foetal lungs is in part due to the release of mir17~92 cluster. funding: cihr-sickkids foundation grant. bottom-up assembly of fully-synthetic extracellular vesicles oskar staufer a , franziska dietrich a , jochen hernandez a , martin schröter a , sebastian fabritz b , heike böhm a , ilia platzman a and joachim spatz a a max planck institute for medical research, department for cellular biophysics, jahnstraße 29, 69120 heidelberg, germany, heidelberg, germany; b department for chemical biology, max planck institute for medical research, jahnstraße 29, 69120 heidelberg, germany, germany, germany introduction: extracellular vesicles (evs) are considered as key elements for future therapeutic and diagnostic procedures. however, despite enormous research efforts to understand their physiological relevance and several greatly successful clinical trials, evs are currently not authorized for clinical routines by american or european regulation and approval agencies. this is especially because therapeutic evs are produced or isolated from cell cultures or biofluids, both of which are subjected to batch-to-batch variations and ill-defined contaminations. therefore, complementary technologies that produce evs as reproducible and defined as state of the art nanotherapeutics, would revolutionize the application of evs in clinical settings and provide the scientific community with a holistic understanding of ev-mediated signalling processes. in our study, we achieve de novo bottom-up assembly of fully synthetic evs (fsevs) that comprise identical physiological and therapeutic functionalities to natural evs. methods: we applied droplet-based microfluidic synthesis to sequentially amalgamate synthetic lipids, proteins and nucleic acids into defined vesicles that display analogous therapeutic capabilities to natural evs. fsevs were characterized by electron and confocal microscopy, dynamic light scattering and mass spectrometry and tested on organotypic models or in vivo. results: using previously described evs as "naturegiven" blueprints, we assembled several fsevs in their exact molecular composition. in particular, we produced wound-healing promoting evs composed of several exosomal proteins, lipids and micrornas and showed that their therapeutic performance on human skin wounds is equivalent to that of natural evs. besides their high molecular complexity, being composed of dozens of different molecular building-blocks, the presented fsevs are completely defined on a quantitative level. based on this, we achieved a stoichiometric understanding of cell-vesicle interactions. summary/conclusion: by applying bottom-up synthesis of fsevs for quantitative studies on ev signalling, we not only provide innovative and safe compounds for ev-therapeutics but also a vastly new perspective on the application spectrum of extracellular vesicles in fundamental research. introduction: small extracellular vesicles (sevs) contain functional molecules from their cell of origin and can enter recipient cells for intercellular communication. ifnβ has been shown to induce some lncrnas to regulate host immune response and play a major role in the positive regulation of the activity of natural killer (nk) cells. here, we aim to clarify whether ifnβ induced sevs can regulate the cytotoxicity of nk cells by transferring specific lncrnas into nk cells. methods: evs were purified from a549 with/without ifnβ treatment by serial centrifugation followed by sucrose density gradient purification. elisa assay were performed to demonstrate the cytotoxicity of nk cells. qpcr and western blot were used to verify the expression of nkp46. results: surprisingly, ifnβ induced sevs can strengthen the cytotoxicity of nk cells. through human transcriptome array (hta) we found the expression levels of 69 lncrnas were significantly changed within sevs isolated from a549 cells following ifnβ treatment. additionally we found a specific sev cargo, linc-epha6-1, acted as a competing endogenous rna (cerna) for hsa-mir-4485 which subsequently up-regulate the natural cytotoxicity receptor (nkp46) expression. furthermore, we verified over-expression of linc-epha6-1 significantly enhance the cytotoxicity of nk cells against zika virus-infected a549 cells. summary/conclusion: our results demonstrated that ifnβ-induced linc-epha6-1 wrapped in sevs can regulate the cytotoxicity of nk cells. our study provides a novel link between type i ifn and nk cells, which are two major players for the host innate immunity against pathogen infections. introduction: hiv-infected t cells release simultaneously viral particles and small extracellular vesicles (sevs) including mvb-derived exosomes and plasma membrane-derived evs. sevs and hiv share many physical and chemical characteristics, which makes their separation difficult. although several approaches have been used to obtain sevs free of virus they leave a majority of sevs within hiv preparations. for this reason, the function of sevs during hiv infection remains unclear. methods: we have developed a novel un-biased proteomic profiling approach to identify specific markers of the virus or sev subtypes released by a human t lymphoma cell line. our approach was to combine differential centrifugation of medium/small evs contained in the ccm with quantitative mass spectrometry to generate protein abundance profiles across the different sub-fractions. we generated an interactive database to define groups of proteins with similar profiles, suggesting their release in the same evs. results: we thus identified different categories of evs, which bear different surface proteins, e.g. different combinations of t cell surface markers, integrins or tetraspanins. in evs released by infected cells, we identified cellular proteins behaving like hiv proteins, and several that changed behaviour after infection, either moving towards or away from the hiv cluster. we identified two cell-derived proteins that are included in the viral particles and one that is specific of non-viral sevs that are modified by infection, and analysed their respective roles in controlling ev composition or virus infectivity. summary/conclusion: our approach presents a powerful tool for identification of common cargoes of given ev subtypes, and could be now used to identify modifications of ev composition in any given physiological or pathological situation. the encephalomyocarditis virus leader modulates autophagic pathways to promote the release of virions inside extracellular vesicles introduction: recent data indicate that naked viruses belonging to the picornaviridae family can be released from host cells via enclosure in extracellular vesicles (ev). ev cloak virus particles in a host-derived "envelope" and can thereby affect antiviral immune responses and disease severity. a better understanding of the formation and function of ev-enclosed viruses is therefore required. previously, we showed the presence of the autophagosome marker lc3 in ev isolates from encephalomyocarditis virus (emcv) infected cells, suggesting the involvement of a secretory autophagy pathway in ev-mediated virus release. however, little is known about the viral and host factors that regulate this process. here, we have assessed the role of the emcv leader, a viral protein that is dispensable for replication but is required for symptomatic disease. methods: cells were infected with wildtype virus or a mutant carrying an inactive leader. ev produced during the infection were isolated using differential ultracentrifugation and density gradient purification. ev were characterized by high resolution flow cytometry and their infectivity determined using end-point dilution assay. in addition, the fate of autophagosomes in infected cells was monitored using a reporter assay for autophagosome-lysosome fusion and analysis of the secretion of autophagosomal proteins. results: inactivation of the emcv leader strongly reduced the release of ev-enclosed virus. whereas autophagosomes are typically degraded, we show this is blocked by the leader. instead, autophagosomes fuse with the plasma membrane, as indicated by the secretion of autophagy marker lc3 during infection with wildtype but not the mutant virus. pharmacological reactivation of degradative autophagy in infected cells resulted in a strong reduction in the release of ev and ev-enclosed virus. similarly, the reduction in evenclosed virus release in the absence of the leader could be partially reversed by drugs that promote the secretion of autophagosomes. summary/conclusion: our data supports a role for secretory autophagy in the release of viruses in ev, a pathway that is regulated by the emcv leader. these findings highlight an unconventional route for ev formation that intersects with autophagosomal compartments and contributes to viral pathogenesis. introduction: zika virus (zikv) causes a public health emergency of international concern because of its correlation with microcephaly. during viral infection, the innate immune response quickly to produce some endogenous functional molecules which can prevent viral invasion or replication. extracellular vesicles (evs) contain molecules from their cell of origin under virus infection and can enter recipient cells for intercellular communication. here, we aim to clarify whether zikv induced evs can regulate viral pathogenicity by transferring specific rna. methods: evs were purified from a549 with/without zikv infection by serial centrifugation followed by sucrose density gradient purification. human transcriptome array (hta) was used to found rna expression within evs. flow cytometry was used to determine cell cycles. zikv replication was assayed by qpcr and western blot. flow cytometry was used to determine cell cycles. results: through hta we found the defensin alpha 1b (defa1b) expression was significantly increased within evs isolated from zikv infected a549 cells. additionally, we found that the extracellular defa1b but not the intracellular defa1b exerts anti-zikv effect mainly before entry step. surprisingly, up-regulate defa1b can retard cell cycles of host cell. we verified defa1b could bind with the origin recognition complex 1 (orc1) which is required to start dna replication during the cell cycle. furthermore, up-regulate defa1b decreased the orc1 level in nuclear. interestingly, evs with defa1b can internalize into recipient cells and inhibit their cell cycles. summary/conclusion: together, our results demonstrated that zikv infection can induce defa1b wrapped in evs, and defa1b not only exerts anti-zikv effect but also regulate cell cycles which may affect neurodevelopment. our study provides a novel viewpoint that defa1b act as first-line anti-viral molecules during zikv infection also correlate with neurodevelopment by retarding cell cycles. extracellular vesicles mediate bacterial-immune cell interactions during respiratory viral-bacterial co-infections sidney w. lane a , matthew hendricks b and jennifer bomberger a a university of pittsburgh, pittsburgh, usa; b university of washington, seattle, usa introduction: respiratory infections are a major cause of morbidity and mortality worldwide and host-derived extracellular vesicles (evs) play important roles in mediating these infections. during respiratory infection, evs are shown to have a modulatory effect: promoting or suppressing infection dependent on the pathogen and cell type. in the age of next-generation sequencing, we now appreciate that many respiratory infections are polymicrobial in nature, with viral-bacterial co-infections correlating with worse disease outcomes. epidemiological studies correlate acute viral infections with the increased likelihood and severity of both acute and chronic secondary bacterial infections; however, the exact mechanisms of these interactions remain poorly understood. evs have been understudied in the context of respiratory viral-bacterial coinfections; thus, their role in mediating these infections is relatively unknown. unpublished data from the lab shows that in airway epithelial cells (aecs), viral infection induces the release of evs that associate with pseudomonas aeruginosa (pa) and promote biofilm growth. here, we aim to expand upon these findings and determine how aec evs mediate pa-immune cell interactions during respiratory viral-bacterial co-infection. methods: to determine how exposure to evs impacts pa-immune cell interactions, evs were isolated from the apical secretions of aecs and co-cultured with pa. ev-treated pa was then co-cultured with macrophages to evaluate ev impact on pa uptake and survival. results: in preliminary experiments using control evs, we observed that evs associate with pa. interestingly, during co-culture with macrophages, ev-treated pa are more susceptible to phagocytosis in comparison to non-treated pa. however, after 4 hours of co-culture with macrophages, ev-treated pa are able to survive and replicate, while nontreated pa are effectively controlled by the macrophages. summary/conclusion: these findings suggest that while pa-ev association promotes pa uptake, it may ultimately enhance pa immune evasion and survival. ongoing experiments in the lab are evaluating the mechanism of pa-ev association and how evs from virus-infected aecs affect the phenotypes observed with control evs. notably, this is one of few reports of a mammalian ev influencing the pathogenesis of a bacterium; thus, results from these experiments will define the function of aec evs in regulating bacterial-immune cell interactions during respiratory co-infections. using machine learning with neuronal ev target proteins and clinical data to predict cognitive impairment in hiv infection lynn pulliam a , michael liston b , bing sun c and jared narvid d a university of california, san francisco, san francisco, usa; b veteran affairs, san francisco, usa; c ncire, san francisco, usa; d ucsf, san francisco, usa introduction: objective biomarkers are needed to assess and predict neuronal function and cognitive impairment. in people ageing with chronic infections, such as hiv, determining the mechanism of impairment will be important when therapies are available. methods: sixty plasma samples from hiv-infected people were obtained from nih-sponsored aids banks. clinical and epidemiological data were collected. all underwent neuropsychological testing and 40 were considered impaired. neuronal extracellular vesicles (nevs) were isolated from plasma and assayed for high-mobility group box 1 (hmgb1), neurofilament (nf-l) and phosphorylated tau-181 (p-tau) proteins. results: using 3 different algorithms, support vector machines (svm) performed the best with an area under the curve (auc) value of 0.82 ± 0.16. using 4 different combinations of clinical data and the 3 nev protein targets, selected clinical data and hmgb1 best predicted cognitive impairment (auc = 0.82). the most important features included cd4 count, hmgb1, nf-l and education. summary/conclusion: specific clinical features plus nev hmgb1, an inflammatory marker, were the best predictors of cognitive impairment. previous published data showed nev p-tau-181 elevated in alzheimer's disease and in this study p-tau had no importance in assessing hiv-associated cognitive impairment. nev target discovery can be improved to better identify neuronal damage, possibly to differentiate other neurodegenerative diseases and hopefully recovery after therapies are identified. in recent years, we have been able to separate and characterize extracellular vesicles (evs) from several different viruses including hiv-1, htlv-1, rift valley fever virus and ebolavirus. however, to date it is not clear whether there is a timing difference between ev and virus release from infected cells. methods: ev isolation by nanoparticle capture and differential centrifugation, ev quantification by nanoparticle tracking analysis, western blot, rt-qpcr, virus rescue assay. results: we have attempted to address the kinetics of ev and virus release from multiple-infected cells using serum starvation experiments from infected (100%) cells. these infected cells were initially put in g0 quiescent stage using serum starvation. both supernatants and cell pellets were collected postinduction release (20% fbs + pma/pha) at 0, 3, 6, 12, and 24 hours and examined for the presence of ev, autophagy and viral proteins as well as viral rna expression. results from supernatants of uninfected cells showed a peak of tetraspanin proteins (cd63, cd81, and cd9) at 6 hours and a gradual decrease of all ev associated proteins by 24 hours. however, the ev from hiv-1 infected cells showed all three tetraspanins present at 3 hours and expression gradually increased up to 24 hours. when compared to htlv-1 infected cells, the three tetraspanin proteins peaked at 6 hours and expression continued to decrease up to 24 hours. htlv-1 infected cells also showed a unique pattern of cd81 expression. autophagy associated proteins (lc3a, lc3b and p62) from uninfected cells and htlv-1 infected cells plateaued at 6 hours, whereas in hiv-1 infected cells their expression continued to increase and peaked at 24 hours. hiv-1 viral proteins (p24, gp120, nef) expression was present at 6 hours and continued to increase and peaked at 24 hours. htlv-1 proteins (p19 and gp46/61) peaked at 6 hours and gradually decreased overtime. hiv-1 and htlv-1 rna gene expression analysis was performed, and data correlated with viral protein expression. additionally, evs release was quantified and showed significant increase of ev concentration overtime in both uninfected and infected samples. finally, experiments of infectivity from 6-and 24hour supernatants were performed on three naive cells. hiv-1 supernatant 6-hour sample was found not to be infectious. however, hiv-1 was successfully rescued from 24-hour sample. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = 31). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and 10 ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine 37 distinct uev surface markers of cd9+/cd63+/cd81+ vesicles in a multiplexed bead-based manner including respective controls. the accuri c6 (bd) was utilized for data acquisition. for further misev2018-compliant characterization, cd9+/cd63+/cd81+ uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd209, all other 36 surface markers could be identified. the most abundant markers were cd9 and cd63, which were detected in 93% of samples, followed by cd133/1 (84%), cd326 (81%), cd81 and cd24 (77%, each). cd3 (42%), cd9, cd45 (39%), cd49e (32%) and cd81 showed similar relative median fluorescent intensities (rmfi), while cd63 yielded significantly higher (p = 0.009) and all other markers significantly lower rmfi (p < 0.011). tem and f-nta revealed cup-shaped vesicles (137 ± 22 nm) with 8.8 ± 7.0 e + 10 particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg101, cd9, cd63 and cd81 without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a ,esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = 40) aged 40-70 yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a 3-colour panel, which included annexin v (for the majority of circulating evs), cd41 (for platelet-derived evs) and cd105 (for endothelial-derived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and 10-yr cvd risk detected by qrisk2. results: ev numbers, as determined by nta, were strongly associated with bmi (r = 0.602, p < 0.001), blood pressure (systolic bp: r = 0.359, p = 0.023; diastolic bp: r = 0.550, p < 0.001) and plasma triacylglycerol levels (r = 0.703, p < 0.001). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = 0.330, p = 0.038). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining 49.4% of the variance for total ev numbers. an additional 9.3% of the variance in total ev numbers was predicted by diastolic bp. ancova of the 10-yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with 10-yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd4 + t cells were isolated from secondary lymphoid organs of foxp3-rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th1, th2, and th17), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, 15-day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for 5 days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th1, th2, th17) showed either no change in proliferation (th1) or decrease in proliferation (th2, th17), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < 0.0001). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th1), il4 and il13 (th2), or il17a and il17 f (th17). in contrast, exposure of tregs to cdc-evs resulted in~1000-fold increase in expression of il10, a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il-10 in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il-10. this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t32hl116273. prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from 42 patients treated with prostanoids (study group; n = 42, 50 ± 16 years, 70% female) and 38 patients not treated with prostanoids (control group; n = 38, 55 ± 19 years, 65% female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; 0.5 mm), adenosine diphosphate (adp; 6.5 µm) and thrombin receptor-activating peptide (trap; 32 µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd61+ and cd62p+; pevs), leukocytes (cd45+, levs) and endothelial cells (cd146+, eevs) were measured in plateletdepleted plasma by flow cytometry (a60-micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = 0.01) and lower concentrations of pevs and levs (p ≤ 0.05). furthermore, thrombus formation was delayed (p ≤ 0.003) and thrombus size was decreased (p = 0.008) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd61+ pevs (p = 0.04). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ 0.05) and the concentrations of pevs (p ≤ 0.08). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ 0.04). introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from 17 cf children (<6 years of age). for nanoflow cytometry, evs were stained with di-8-anepps, and with epcam, cd66b and cd115 (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. results: the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = 0.003, spearman's rho = 0.689). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = 0.001, rho = 0.857). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv15a0), emory pediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: 142 individuals were enrolled into our study, subdivided into a training (volunteer n = 27, pneumonia n = 12, sepsis n = 28) and testing cohort (volunteer n = 20, pneumonia n = 18, sepsis n = 37). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq2) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative realtime pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed 29 significantly regulated mirnas in pneumonia compared to volunteers, and 25 mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by 12 mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: 0.982, pneumonia: 0.965, sepsis: 0.992). mir-193a-5p (log2fc = 1.86, padj = 1.49e-6) and mir-542-3p (log2fc = 1.67, padj = 3.29e-5) differentiated between pneumonia and volunteers and mir-1246 (log2fc = −2.41, padj = 1.78e-04) between pneumonia and sepsis. expression levels of mir-193a-5p and mir-1246 were related to disease severity. mir-542-3p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was 73.33%. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study,3healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd63 and cd81 were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and 9 mirnas (mir-103a-p, mir-191-5p, mir-320a, mir-200b-3p, mir-185-5p, mir-223-3p, mir-21-5p, mir-155-5p, let-7 g-5p) , were evaluated by rt-qpcr. after the optimization of the methodology, 10 healthy adults subjects and 10 patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd63 and cd81. however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna-320a showed a significant increased (p = 0.02) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir-320a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf2-active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds joseph kuhn a , absara hassan b , sonali sharma b , jennifer kwong b , montaha rahman b , salma adam b , jasmine lee b , alvaro villarreal ponce b and piul rabbani b a nyu langone health, new york, usa; b nyu langone health, new york, usa introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid 2-related factor 2 (nrf2) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf2 in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf2-active exosomes, mscs were incubated with potent nrf2 activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express >95% positive msc markers (cd44, cd73, cd90, and cd105) and <5% express negative markers (cd45, cd31, cd14, cd19, or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd81, cd9 and tsg101. nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of 168.0 ± 6.5nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf2-active human mscs increase exosome secretion by 54%, compared to nrf2-baseline mscs (p < 0.05). both nrf2-baseline and nrf2-active exosome treatment significantly reduced closure time to 15.5 and 14 days respectively, compared to 29.8 days for vehicle-treated wounds (p < 0.05). this reduction eliminated the delay in closure time compared to wounds of c57/b6 mice. nrf2-active exosome treatment of db/db wounds reduced closure time by a further 2.6 days compared to untreated c57/b6 wounds. at day 10, exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd31+ vessels compared to vehicle-treated wounds. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist1 is a key mediator of tumour cell metastasis.. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist1. methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc3-ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc3-ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc3-ml lines stably overexpressing or deficient in twist1. results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc3-ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc3-ml overexpressing twist1 showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist1 expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. funding: american heart association of12 symposium introduction: as researchers continue to explore the therapeutic potentials of extracellular vesicles (evs) for the treatment of many diseases, there is a growing unmet need for real-time in vivo monitoring of these therapeutic evs after they are injected into a subject to understand their safety, targeting, and effectiveness. while current optical imaging solutions like bioluminescence and fluorescence are useful for ev tracking studies in animal models, there is limited utility in clinical applications. here we present a novel ev tracking solution utilizing clinically applicable mri technology. methods: to generate trackable evs, cells were labelled with a clinically applicable novel magnetic agent. evs secreted by the labelled neural stem cells and amniotic fluid stem cells (afscs) were isolated by differential ultracentrifugation. the viability and morphology of labelled-cells were evaluated, and the in vitro mr properties of their derived evs were analysed by magnetometer. a proof of concept in vivo biodistribution study was conducted by injecting labelled evs into wt and alport mice (a model of chronic kidney disease) via retro-orbital and intra-cardiac routes and tracking them via mri at 10 min and 3 hr postinjection. results: the magnetic label did not affect the physiological characteristics of the cells. the mr detectability of labelled-evs was confirmed by in vitro/ex vivo mri phantoms. mri studies showed that homing of afsc evs to the kidney injected intra-cardiacally into alport mice were more efficient versus the retro-orbital route, and prussian blue staining of kidney sections confirmed the mr findings. introduction: a central question in ev biology is the fate of circulating ev. this can be evaluated by developing non-invasive ev bioimaging techniques in mice in order to benefit from transgenic and knock-out models. recent reports described ev biodistribution in vivo using optical (fluorescence) and nuclear imaging. but the physicochemical properties of the probes impact ev integrity, labelling efficiency, background signals and observation timecourse. methods: we developed the radiolabeling of red blood cells (rbc) and ev with [18 f]fluorodeoxyglucose (18 f-fdg). we used rbc-derived ev in their native, intact form, without pre-experimental processing (no centrifugation or filtration). we tracked 18 f-fdg in vivo by pet-scan, within seconds of ev, rbc or free 18 f-fdg injection, and during their dissemination in blood and recruitment by organs over one hour. ev and rbc biodistribution were confronted to the kinetics of free 18 f-fdg. results: we collected images of the biodistribution of rbc, and rbc-derived ev. nuclear imaging was well suited for accurate studies of ev organotropism, with high sensitivity, excellent signal-to-noise ratio, very low signal absorption by tissues and an inherent quantitative tomographic nature. ev-specific signals were mostly accumulated within minutes of injection (tail vein), in the spleen and liver, with a small part in the bone marrow (femurs). signals in other compartments were largely transient and linked to tissue perfusion and blood volume. we selected the most drastic control conditions to secure a correct interpretation of the data. this made kidneys, hearts and brains unavailable for analysis. hence the new approach came with limitations, but we describe how "free" 18 f-fdg signals can be used to draw sound conclusions for ev. summary/conclusion: we propose that three types of compartments coexist in control mice at rest: active ev-capturing organs with high capacity and specificity including the spleen, and to a lesser degree the bone marrow; passive ev-retaining organs with high capacity, including the liver; and ev-neutral organs where transient signals only mirror tissue perfusion. we also report how ev biodistribution patterns are altered in ageing animals, as an example. we hope that this novel, non-invasive, quantitative, dynamic wholebody imaging approach will help characterize native cell-derived ev and help set standards for the reproducibility of ev bioimaging in mice. funding: frm grant "biface", inserm copoc, cnrs. introduction: extracellular vesicles (ev) are important mediators of intercellular communication; however, basic principles of ev biogenesis and loading remain largely unknown. a limited repertoire of tools has thus far made these processes challenging to research. the development of an ev-transfer reporter in a genetically tractable organism such as drosophila has allowed us to study mechanisms of cargo loading in vivo and has provided us with a platform to explore fundamental aspects of ev biology. methods: we have developed a bioinformatic pipeline to analyse the properties embedded in the 3ʹutr of mrnas enriched in evs released by drosophila cells. in parallel, we have adapted a cre-loxp system for use in fruit flies that appears to be proficient to reveal the exchange of bioactive molecules between secretory and recipient cells. results: taking advantage of computational methods, we uncovered sequence motifs that preferentially appear in combinations along the 3ʹutr. these sequence motifs occur within characteristic secondary structures, in a way that is more variable and motif dependent than previously reported. identified motifs also show similarities to known binding sites for rna binding proteins; a feature potentially important for ev-loading. in parallel, we developed a drosophila in vivo system to detect cell communication in complex tissues and between different cell types. using this system, we studied the biological significance of specific sequence motifs and identified their ability to modulate mrna ev-transfer in a context dependent and evolutionarily conserved manner. summary/conclusion: in summary, we have developed a novel tool to study cell communication in complex tissues, and shown its effectiveness to study principles of ev biogenesis and loading. beyond improving our understanding of ev biology and providing a novel tool to the scientific community, we hope this knowledge will pave the way to harnessing evs as a means of remotely manipulating cell communication in many biological contexts. introduction: the idea of cross-kingdom, species and inter-individual transfer of bioactive compounds via extracellular vesicles (evs) is a recent avenue. however, the bioactivity and bioavailability of these dietary compounds upon consumption is highly debated. it has been proposed that evs from diet can absorbed by consuming organisms, be bioavailable in various organs and exert phenotypic changes. milk is the most vastly consumed beverage and is an abundant source of evs that may act as signalosomes. whether these milk-derived evs can serve as cross-species messengers and have a biological effect on host organism has been poorly understood. methods: bovine milk-derived evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. the evs were characterised by tem, nta, quantitative proteomics and rna-seq. evs were orally administered to various mice models of colorectal, breast and pancreatic cancer. primary tumour burden was monitored, and the rate of metastases was measured by imaging and qpcr. immune cells were analysed by facs. mechanistic insights were obtained using quantitative proteomics, confocal microscopy and biochemical experiments. results: we demonstrated that upon oral administration, bovine milk-derived evs were able to survive the harsh degrading conditions of the gut and be bioavailable in peripheral tissues. interestingly, oral administration of milk-derived evs reduced the primary tumour burden in various cancer models and attenuated cancer cachexia. intriguingly, despite the reduction in primary tumour growth, milk-derived evs accelerated metastasis in breast and pancreatic cancer mice models. timing of ev administration was critical as oral administration after resection of the primary tumour reversed the pro-metastatic effects of milkderived evs in breast cancer. biochemical and quantitative proteomics analysis highlighted the induction of epithelial-to-mesenchymal transition and senescence upon treatment with milk-derived evs. summary/conclusion: taken together, we were able to demonstrate the capacity of bovine milk-derived evs in mediating cross-species communication and regulating cancer progression in a context-dependent manner. bacterial membrane vesicles (mvs)a bacterial innate defence system against viral infection xiaomei yan, qian niu and ye tian xiamen university, xiamen, china (people's republic) introduction: in order to survive the constant onslaught of phage, bacteria have evolved diverse defence mechanisms that act at every stage of the phage life cycle. it has been suggested that bacterial membrane vesicles (mvs) may play a key role in innate bacterial defence against phage infection by acting as a decoy to prevent phage adsorption. nearly a decade has passed since mvs were first proposed as a decoy, but details of how bacteria utilize mvs to defend against phages remain poorly understood. here we use the laboratory-built nano-flow cytometer (nfcm) to reveal details of the interaction between mvs and phages at the single-particle level, and to provide new insights into innate defence mechanisms of mvs. methods: s. typhimurium was used as the model system. differential ultracentrifugation and density gradient centrifugation were used to isolate and purify mvs and bacteriophage p22. cryo-tem was used to determine the morphologies of mvs and phage p22. the purity of mv isolates was validated by measuring the particle concentration before and after triton x-100 treatment. monodisperse silica nanoparticles were used as the size reference standards to measure the size distribution of mvs via single-particle light scattering detection. the purity of phage p22 was verified by concurrent detection of side scatter and fluorescence signals of single phages upon nucleic acid staining by syto 82. results: by incubating mvs and af532-labelled p22, the number of phages adsorbed on single mvs were accurately quantified. we found that s. typhimurium and mvs it secretes express different affinity for phage p22 attachment. the binding ability of p22 to mvs is greater than that of bacteria. we confirmed that p22 can inject their nucleic acids into mvs, and these nucleic acids can be degraded by non-specific nucleases inside mvs for the first time. besides, by labelling the nucleic acids of mvs with syto 82, we were able to distinguish three different subpopulations of mvs. summary/conclusion: taking advantage of the superior sensitivity of nfcm in single-particle analysis, we developed a novel approach to the characterization of the interaction between mvs and phages. our study revealed that bacteria produce mvs as bait to attract viral adsorption and nucleic acids injection. funding: this research was supported by the national natural science foundation of china (grants 21934004, 21627811 and 21475112). introduction: the development of evs for therapeutic applications requires an in-depth understanding of their in vivo biodistribution and pharmacokinetic profile. in this study, we have made a comprehensive comparison of nuclear, fluorescent, and bioluminescent imaging technologies to identify the most suitable in vivo ev tracking method. methods: evs were purified from expi293 f cell supernatant by differential centrifugation followed by iodixanol density gradient separation and further characterized following misev guidelines. engineered expi293 f cells were used to generate evs carrying mcherry or nanoluc (nluc) proteins. the membrane of naïve ev was labelled with indium111(in111)-dtpa or xenolight dir post-ev isolation. ct26 tumour-bearing balb/c mice were intravenously dosed with 1 × 1011 evs followed by imaging at 1 h, 4h and 24h using spec/ct and ivis systems. tissue distribution and blood circulation profile of evs were analysed from ex vivo samples up to 24h post-injection. results: xenolight dir and (in111)-dtpa were the most suitable ev labels for live whole-body animal imaging, ex vivo organ imaging, and tissue lysate quantification. nluc was appropriate for ex vivo imaging and tissue lysates quantification, but suboptimal for live imaging with limited sensitivity. mcherry evs were found not suitable for in vivo tracking studies due to high background signal fluorescence. ex vivo organ quantification of in111-dtpa and dir showed that naïve evs mainly accumulate in liver, followed by spleen, kidneys, and lungs at 24h post-dose, with less than 5% ev exposure to the tumours. interestingly, nluc-evs accumulated mainly in the lungs, regardless of the small size of the particles injected and the absence of aggregation. blood circulation profile of in111-dtpa and nluc evs showed rapid clearance of vesicles from circulation, with 15% of injected dose detected in blood after 30 min and less than 5% after 16 h. summary/conclusion: radionuclide imaging is an excellent technology to detect evs in vivo and ex vivo with high resolution and sensitivity but requires advanced infrastructure for radiolabeling. the optical methods have limited tissue penetration and sensitivity but can be improved with the right selection of the dye. these results contribute to the understanding of the biodistribution and pharmacokinetics of evs and are highly relevant to exploiting their potential for targeted delivery to diseased tissues in vivo. symposium introduction: new methods for quantifying extracellular vesicles (evs) in complex biofluids are critically needed. we report the development of a new technology combining size exclusion chromatography (sec), a commonly used ev purification technique, with fluorescence detection of specifically labelled evs (flu-sec). methods: flu-sec was validated using red blood cell derived evs (revs). size and concentration measurements were performed by microfluidic resistive pulse sensing (mrps) using the ncs1 instrument (spectradyne llc, usa). pe-cd235a (anti-glycophorin a) and alexa647-wga (wheat germ agglutinin) were used to label revs. flu-sec experiments were performed on a liquid chromatography system using a tricorn 5/200 glass column filled with sepharose cl-2b gel (ge healthcare). results: a log-normal size distribution was obtained for revs with a mean diameter of 163.5 ± 0.7 nm and standard deviation of 30.6 ± 0.6 nm. the concentration of revs measured by mrps was 3.14*10e11 particles/ ml. the fluorescence chromatograms of the rev samples labelled with pe-cd235a and with alexa647-wga show the typical features of the separation of evs from soluble proteins with sec and enables the determination of the labelling efficiency of the markers. the linear range for quantification of evs in our experiments spans over two orders of magnitude ranging from 10e9 particles/ml to 10e11 particles/ml. the lod depends on the type of the label. in our experiments the lowest lod was 10e9 particles/ml for alexa647-wga. summary/conclusion: the results indicate that flu-sec is a quantitative technique with very good linearity over a wide range of concentrations, though the limit of detection depends largely on the employed label (sci. rep. 9, 19868, 2019) . moreover, the ratio of ev-bound and free-antibody molecules can be also determined by flu-sec, which can be used to calculate the labelling efficiency of the used marker. funding: this work was supported by the national research, development and innovation office (hungary) under grant numbers pd 121326 and nvkp_16-1-2016-0007. zv was supported by the janos bolyai research fellowship. the conan assay: purity grade and concentration of ev microlitre formulations by colloidal nanoplasmonics. (evs). control over such properties is constantly experienced by researchers to be critical for ev proper manipulation, engineering and translation. however, the need for characterization methods that strike the balance between robustness, working volume, cost and accessibility remains unmet. methods: the colorimetric nanoplasmonic (conan) assay we developed consists of a solution of gold nanoparticles (aunps) into which 1-2 μl of the ev formulation is added. the solution turns blue if the formulation is pure, while stays red if soluble exogenous single and aggregated proteins (saps) are present. the colour shift is visible by the naked eye and can be quantified by conventional uv-vis spectroscopy, providing a quantitative index of purity and an estimation the ev molar concentration (particle number). results: the assay specifically targets saps, and not the ev-related proteins, with a detection limit < 50 ng/μl (an order of magnitude higher resolution than the bradford protein assay). for pure solutions, the assay also allows for determining the ev number, as the colour shift is linearly dependent to the aunp/ev molar ratio. instead, it automatically reports if the solution bears sap contaminants, thus avoiding counting artefacts. experiments, conducted on ev separated from milk and ascaris suum culture medium, are repeatable, with an error below 20%. summary/conclusion: conan proves to be robust and reliable, while displaying appealing performances in terms of cost (inexpensive reagents, run by standard microplate reader), working volumes (1-2 μl) and time (the procedure takes less than one hour). the ability to assign a quantitative purity grade is, up to date, a unique peculiarity of this assay. finally, the assay is potentially extendable to all classes of natural and artificial lipid micro-and nanoparticles. funding: evfoundry project, horizon 2020 -future and emerging technologies (h2020-fetopen), id: 801367. marina cretich a , roberto frigerio b , alessandro strada b , greta bergamaschi b , marcella chiari c and alessandro gori c a consiglio nazionale delle ricerche (cnr), istituto di scienze e tecnologie chimiche (scitec), milano, italy; b consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy; c consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy introduction: small extracellular vesicles (sev) present fairly distinctive lipid membrane features in the extracellular environment. these include high curvature, lipid packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sev membrane could be then considered as a "universal" marker, alternative or complementary to traditional characteristic surface-associated proteins. here we introduce the use of membrane sensing peptides as new, highly efficient ligands to directly integrate sev capturing and analysis on a microarray platform. methods: we designed and synthesized membranesensing peptide ligands as molecular baits for small ev and we demonstrate their use in a microarray platform as valuable alternative/complement to antibodies. evs from blood serum and plasma were isolated by ultracentrifugation, characterized by tem, nta, wb. samples were analysed by label-free, single particle counting and sizing on peptide microarrays coupled to fluorescence co-localization immune staining with fluorescent anti-cd9/anti-cd63/anti-cd81antibodies. results: peptide microarrays were realized using a click-chemistry strategy for optimal peptide surface orientation and used to analyse evs from human blood. membrane sensing peptides showed a capturing capacity higher than anti-tetraspanin antibodies. in addition to purified vesicles, peptide ligands were tested with pure serum showing capacity to capture evs even from complex samples. in order to get insights into the ev-peptide binding mechanism and verify whether it is directly mediated by the lipid membrane, trypsin-treated evs were captured on peptide microarrays demonstrating that binding is not directly mediated by surface associated proteins. summary/conclusion: we introduced the use of membrane sensing peptides as a novel class of molecular ligands for integrated sev isolation and analysis, reporting for the first time on peptide microarrays for extracellular vesicles. given their affinity to the membrane of small ev, these molecules can serve as general baits, enabling vesicles capturing unbiased by differential surface protein expression. these new class of molecular probes may be integrated with the use of protein markers towards improved small ev isolation and characterization. compared to proteins and antibodies, peptides are characterized by low cost of preparation, remarkable stability and ease of chemical manipulation, offering virtually unlimited possibilities for experimental design. we anticipate that this new class of ligands, may greatly enrich the molecular toolbox for ev analysis. funding: hydrogex (regione lombardia&fondazione cariplo, grant n. 2018-1720) and index (european union's horizon 2020 research and innovation programme under grant agreement n°766466) projects are acknowledged for partial financial support. high-resolution size-based profiling and morphological analysis of extracellular vesicles by scanning electron microscopy sara cavallaro a , federico pevere b , petra hååg c , kristina viktorsson c , rolf lewensohn c , jan linnros a and apurba dev b a kth royal institute of technology, stockholm, sweden; b uppsala university, uppsala, sweden; c karolinska institute, stockholm, sweden introduction: extracellular vesicles (evs) have been found to mediate intercellular communication in physiological and pathological processes. nevertheless, the understanding of evs bio-functionality remains elusive, mainly because of their high heterogeneity in molecular content, but also in size (30-2000 nm) . therefore, accurate size measurements of evs are highly desired, particularly for exploiting their full diagnostic/therapeutic potential. currently available techniques, such as nanoparticle tracking analysis (nta), cannot accurately measure evs smaller than 70 nm and are not capable to distinguish them from protein aggregates. on the contrary, electron microscopy (em) techniques allow high-resolution size-profiling and morphological analysis of evs over their whole size range. however, their low throughput combined with several long preparatory steps have prevented em from being routinely used for ev size profiling. methods: we shall present a method improvement in throughput and reproducibility of ev size-analysis by scanning em (sem). the technique is based on covalent ev capture onto a silicon wafer, using the protocol reported by cavallaro et al. up to the glutaraldehyde step. after immobilization, critical point drying (cpd) is performed to dehydrate evs before sem, while preserving their shapes. results: sem images, showing the comparison in densities of evs prepared by covalent and non-covalent coupling to substrate, indicated a good capture efficiency of our covalent protocol. the size distribution analysis showed good agreement between nta and sem for evs >80 nm. for smaller evs, sem is more sensitive than nta, thus more suitable to check the purity of ev-isolation techniques. last, atomic-force microscopy (afm) measurements was also used to validate our measurements. introduction: extracellular vesicles (evs) are membrane vesicles secreted into extracellular space, by almost all cellular populations, playing a major role in cell-to-cell communication. it has been already demonstrated that changes in luminal or surface protein cargos of these vesicles, may reflect the status of producing cells. for this reason, evs are considered as potential biomarkers in several types of diseases ranging from cancer diagnosis to heart rejection. periostin (postn) is a matricellular protein associated with evs, and its level is considered a possible biomarker, which indicate malignancy and poor clinical outcome in different types of cancer. here we extensively characterize the presence of postn associated on evs, showing how different isolation methods can drastically affect the amount of postn content in extracellular vesicles fraction. methods: serial ultracentrifugation steps or size exclusion chromatography were used to isolate evs from primary culture of cardiac progenitor cells. evs were characterize, according to misev guidelines, by western blot, nta, facs and cryotem analysis methods. postn amount, associated with evs, was analyses by western blot and elisa. furthermore, functional tests were performed on h9c2 cardiomyoblast cell line, treated with the same amount of evs from different isolation methods; cells response were analysed by western blot. results: evs, from both the isolation methods, showed tsg101, syntenin1, cd63 positivity while grp94 was absent. nta showed no differences, in terms of amount and size of particles. by facs analysis evs resulted enriched with cd63, cd9 and cd81. cryotem showed a similar morphology in the two preparations with presence of protein contaminant in the ultracentrifuge pellet. in vitro, h9c2 treated with evs showed activation of pfak after 10ʹ of treatment, this induction was 1.5 times higher in cells treated with evs isolated with ultracentrifuge compared to evs isolated with sec, confirming a drastic effect of postn protein contamination. furthermore, by phospholipase-c treatment, we found that postn is bound to evs surface through a gpi anchor. summary/conclusion: these results suggest that selection of a proper isolation method is critically relevant in evs studies, in particular when protein analysis is considered. different isolation methods dramatically influence protein amount in extracellular vesicles and consequentially their function. furthermore, in this study we show for the first time, that postn is actually bound to evs surface and not carried in their lumen as previously believed. members of the y-rna family have been detected in ev from various cell types and are among the most abundant non-coding rna types in plasma. we previously showed that shuttling of full-length y-rna into ev is modulated by tlr-activation of ev-producing immune cells. this suggested that y-rnas may have potential as biomarker for immune-related diseases. methods: we separated rna-containing structures in plasma based on differences in size, density, and resistance to protease/rnase treatment. using rt-qpcr, we quantified full-length y-rna subtypes (y1, y3, y4) in ev from various blood-related cell types cultured with or without lps-stimulation. inflammationinduced changes in y-rna were assessed in plasma samples from a human endotoxemia study. results: full-length y-rna in plasma was mainly found in ev (early sec-fractions, density 1.11-1.18 g/ml). in contrast, specific mirnas were either enriched in lpp (e.g. mir-122), in both ev and lpp (e.g. mir-16 and mir-21), or in ev (e.g. mir-150). evenclosed full-length y-rna was resistant to enzymatic degradation, while lpp-bound mirnas were degradation sensitive. we discovered that ev released by different blood cell types varied in y-rna subtype ratios. these ratios remained stable upon lps-stimulation of the ev-producing cells. in endotoxemia plasma samples, the neutrophil-specific y4/y3 ratios and pbmcspecific y3/y1 ratios changed significantly during systemic inflammation. importantly, the plasma y-rna ratios strongly correlated with the number and type of circulating immune cells during the inflammation process. summary/conclusion: cell type specific "y-rna signatures" in plasma ev can be determined without prior ev-enrichment, and may be further explored as biomarkers to diagnose inflammatory responses or other immune-related diseases. mining public ev small rna-seq data with mirev -insights into potential reference transcripts and abundant mirnas recently, extracellular membrane vesicle (mv) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells. s. aureus produce membrane-bound, spherical, nano-sized, mvs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. the present study sought to examine the nature of the association between nucleic acids and mvs produced by s. aureus. we also sought to analyse the immunostimulatory potential of mvassociated rna and dna, and to evaluate receptormediated recognition of mv-associated rna and dna molecules by innate immune cells. methods: by following a stringent purification protocol, we characterized the rna and dna content of mvs produced by actively growing s. aureus. nuclease protection assays were performed to determine whether mv-associated nucleic acids are protected from degradation. we assessed the immunomodulatory potential of mv-associated rna and dna by treating cultured mouse macrophages with mvs and measuring the induction of interferon-β mrna using qpcr. introduction: urinary extracellular vesicle (uev) transcriptome could potentially reflect the kidney gene expression profile and serve as virtual/liquid biopsy. in order to explore this possibility, we performed mrna sequencing of uevs from individuals with type 1 diabetes to assess whether it can capture a "kidney enriched genes" expression signature that could lead to novel biomarker discovery for diabetic kidney disease. methods: the study included 75 type 1 diabetic individuals (41 normoalbuminuric, 15 microalbuminuric and 19 macroalbuminuric). urine samples were collected either overnight (n = 38) or during 24-hours (n = 37) and uevs were isolated from 20-30 ml of urine by differential centrifugation. the evs quality was ensured by electron microscopy (em), western blotting and ev-rnasprofiling with the bioanalyzer. isolated rnas were subjected to rna sequencing after cdna library preparation (ultra-low amount protocols) using hiseq 2000 (illumina) pair-end protocol. the association between kidney specific gene expression levels (>4 fold higher compared to other tissues, n = 53) and degree of albuminuria or glomerular filtration rate was explored. results: isolated ev quality appeared good by em and western blotting. rna quantity and quality were sufficient for sequencing of all samples with >5 million pair end reads. we detected on average expression of 12,642 genes. principal component analysis (pc) of the expression of all genes did not reveal any systematic batch differences between the overnight and 24-hour urine collections. comprehensive look-up of 53 kidney-enriched genes revealed expression of >73% (total 39) of these genes in urine evs with high expression of five kidney-specific genes (slc12a3, slc12a1, nphs2, aqp2 and slc22a12). pc analysis combining the impact of 39 kidney-enriched genes revealed that most macroalbuminuric patients clustered together along the pc1 axis, and the axis also correlated with the albumin-to-creatinine ratio (p = 0.0004) explaining 11% of the variance (p = 0.005) in the whole data set. the pc1 axis also showed correlation with hba1c (p = 0.003), but not with diabetes duration, bmi, age and egfr. introduction: due to their safety profile, tissue tropism and long-term transgene expression, adeno-associated viruses (aavs) have become the vector of choice for human gene therapy. however, pre-existing neutralizing antibodies (nabs) to many aav serotypes pose a critical challenge for the translation of gene therapies to clinic. here, we describe the use of exosomal aavs (eaav) as a robust cardiac gene delivery system that enhance transduction efficiency while shielding from pre-existing humoral immunity to the viral capsid. methods: we developed an ultracentrifugation-based purification strategy to obtain eaav specimens from aav-producing hek-293 t cells, and used electron microscopy-based visualization, confocal microscopybased colocalization studies, qpcr, immunoblotting, dynamic lights scattering, exoview technology and protease assays to characterize eaav morphology, contents and mechanism of action. we then evaluated efficiency of heart targeting for eaav9 or eaav6 and standard aav9 or aav6 encoding for egfp, mcherry or firefly luciferase in different human cell lines in vitro, in black mouse and in passive immunity nude mouse model in vivo using flow cytometry, confocal microscopy, langendorff perfusion system and methods: hlhs patients (n = 3) after glenn procedure and swine (n = 3) after pab were given rv injections of allogeneic/xenogeneic mscs. donor-specific, hla-i+, exosomes were isolated from plasma. in swine, exosomes were collected and rv fractional area change (fac) was measured post-msc-injection. in the elpis patients, exosomes were collected and outcome measurements (fac, stroke volume (sv), rv mass) were recorded 6 and 12-months post-injection. exosomal mrna, microrna (mirna), and proteins were quantified and partial least squares regression (plsr) reduced the dimensionality of the datasets to build a swine model, upon which elpis outcome predictions were made. results: multiomics analysis of swine exosome cargo revealed mirna to be the largest contributor to overall variance. in swine and elpis patients, mirnas were similarly expressed (98%, fold-change<2). plsr reduced the dimensionality of the swine mirna dataset to 50 mirnas with the highest weighted coefficients for changes in fac. pathway analysis of mirna targets revealed links to smooth muscle cell proliferation and cardiac chamber development. importantly, the swine mirna plsr model predicted elpis patient improvements in fac, sv, and rv mass with strong correlation (r > 0.9). summary/conclusion: these findings support the use of: (1) swine pab model for rv failure in hlhs, (2) circulating donor-specific msc-exosomal mirna as a novel, non-invasive biomarker of patient outcomes, and (3) introduction: evs have been shown promising potential as a drug delivery vehicle, especially nucleic acid therapeutics. however, the overall short of specificity to target cancer cells has led to low therapeutic efficacy and potential toxicity. rna nanotechnology is the bottom-up self-assembly of nanometre-scale rna architectures. we previously discovered a stable phi29 prna three-way junction (3wj) motif and used it to construct multivalent rna nanoparticles with high chemical and thermodynamic stability. the resulting arrow-shape rna nanoparticles are homogenous, uniform in size and shape, and can harbour different functionalities while retaining their tertiary folding and independent functionalities both in vitro and in vivo. this flexible platform using rna nanotechnology to achieve tumour-specific targeting has been demonstrated over the last decade. here we introduce a strategy to take advantage of both evs and rna nanotechnology to develop a versatile platform for efficient target-specific delivery of sirnas for cancer treatment. methods: we design membrane-anchoring arrowtail 3wj rna nanoparticles to display tumour targeting ligand (psma rna aptamer or egfr rna aptamer or folate) on birc5 sirnas loaded evs (fig.1 ). nanoparticles were characterized by nanoparticles tracking analysis (nta), transmission electron microscopy (tem), dynamic light scattering (dls) and atomic force microscopy (afm). evs were produced by hollowfiber bioreactor and purify by tangential flow filtration (tff) follow by ultracentrifugation. cell binding were evaluated by flowcytometry and confocal microscopy and gene knockdown effect were assay by quantity reverse transcription-pcr (qrt-pcr). formulated evs were introduced to tumour (prostate, triple negative breast cancer, colon pdx) xenograft mice by tail-vein injection and evaluate in vivo tumour inhibition. results: 1) we found the orientation of arrow-shaped rna can be used to control ligand display on evs membranes for specific cell targeting. 2) by placing membrane-anchoring cholesterol at the tail of the arrow results in display of rna aptamer or folate on the outer surface of the evs and enhance cancer cell binding and uptake. 3) taking advantage of the rna ligand for specific targeting and evs for efficient cytosolic delivery, the resulting ligand-displaying evs or plant derived evs-like nanovesicles were capable of specific delivery of sirna to cells, and efficiently blocked tumour growth in three cancer models. summary/conclusion: we developed an rna-evs based nanoparticles platform and shown the flexibility for different cancer type treatment. related publications: pi f et.al. nature nanotechnology. 2018 , 13:82. li z et.al. sci rep. 2018 introduction: extracellular vesicles (evs) contain plasma membrane surface markers that provide insights into their cell source. until now, our understanding of the circulating ev-biome has been limited by the lack of celland size-specific ev quantitation methods. we have developed and validated a multiplex nanoscale flow cytometry approach to image cell-and size-specific ev populations using a novel human "ev-lyoplate" with 3 differently coloured monoclonal antibodies per well in a 96 well plate format (n = 242 separate antibodies with isotype, stained pbs, unstained plasma, and quant-beads controls per plate). we hypothesized that platelet poor plasma samples from patients diagnosed with pancreatic cancer would have significantly different ev-biome profiles than screen negative study subjects. methods: study subjects were enrolled and sampled before clinically scheduled endoscopic ultrasoundguided biopsy (eus-fna) procedures to screen patients with symptoms of pancreatic duct obstruction who later had at least two years of clinical follow up, including surgical resection in cases of pancreatic neoplasia (n = 36) or at least one follow up clinic visit to confirm resolution of symptoms. blood samples were uniformly collected, processed, and banked per isev recommended guidelines. uniform machine (facsymphony) settings to standardize light scatter and fluorescence detection were based on commercially available beads (eg. megamix). samples were coded and randomized for testing and results were reported as the mean cell-and size-specific ev events/ul of plasma. results: clinical outcomes confirmed 10 cases of cancer and 26 screen negative controls. principle component analysis suggested that a number of different celland size-specific evs were significantly more common in the cancer cases (adjusted p-value < 0.05, with aucs >0.80), including epcam+/cd63+ events likely from cancer cells and cd41+/cd62p+/cd9+ microvesicles from platelets, among others. summary/conclusion: in this proof of principle study employing an ev-lyoplate design and nanoscale flow cytometry, we could reliably discriminate the ev-biomes in patients with cancer from negative controls. ongoing studies will determine whether these discriminators will be validated in larger cohorts and provide at least noninferior predictive value compared with the current gold standard clinical testing assay (eus-fna introduction: small cell lung cancer (sclc) is an aggressive tumour type, usually metastatic at diagnostic leading to poor overall survival. interestingly, sclc tumours are composed by distinct subpopulations of cells that cooperate as an ecosystem to drive tumour survival. since the subtype of sclc may have prognostic significance, the aim of this study was to identify surface marker proteins as biomarkers of sclc. methods: a linear discriminant analysis (lda) model, implemented in python via sci-kit learn, was used to choose the best 4 markers for distinguishing subtypes. this analysis was based on rna-seq data from a previous study. in order to identify ev-based biomarkers that would identify sclc evs and not normal evs, we excluded from this analysis proteins without a verified transmembrane domain and proteins associated with evs expected to be present in white and red blood cells, and endothelial cells (according to exocarta and vesiclepedia databases). we also prioritized proteins that could be pan markers for sclc and that might have prognostic significance. to validate our findings, we performed western blotting and flow cytometry in sclc cell lines from different subtypes. results: our rna analysis indicated that the best 4 surface markers to distinguish sclc subtypes were ceacam5, fam174a, lrfn1, epha2. immunoblot analysis validated ceacam5 and epha2 but not fam174a or lrfn1. we also found that ncam1, a commonly used sclc marker, only marks some of the subtypes. for further analysis, we chose proteins with antibodies validated for flow cytometry as our chosen biomarker platform. flow cytometry analysis of cd24 is suitable as a pan-sclc marker. however, the expression of non-ne cell lines was decreased compared to rna-seq data. summary/conclusion: protein analysis of ceacam5 and epha2 corresponded to rna-seq data. ncam was not detected as a pan marker for all sclc subtypes. however, we could see cd24 expression in all sclc subtypes, indicating it may be a useful pan marker for sclc. future studies will be performed to validate the expression of other surface markers in cells, purified evs, and plasma of sclc patients. funding: nih u01 ca224276 and nih u54 ca217450. leukobiopsyexploiting extracellular vesicle-mediated leukocyte sequestration of cancer-specific signatures introduction: in cancer, extracellular vesicles (evs) act as a unique exit mechanism for mutant and oncogenic macromolecules (proteins, rna and dna) en route from malignant cells to blood1. while this process has inspired major liquid biopsy efforts, the biology of circulating evs that carry oncogenic mutations (oncosomes)2 is still poorly characterized. it is also unclear what part (if any) of the tumour-related cell free dna (ctdna)3,4, a major liquid biopsy analyte, is linked to circulating evs and what is their fate, receptacles and biological activity. methods: we employed as series of cancer cell lines carrying mutations in major oncogenes (hras, her2, egfrviii). ev-dna was analysed by digital droplet pcr (ddpcr), along with nuclear anomalies in donor cells (dapi, electron microscopy) and transfer of dna to recipient cells of endothelial (huvec, mmbec), astrocytic (nha) or myeloid (hl60) origin. blood underwent fractionation into red blood cells (rbc), white blood cells (wbc), platelets (plt), evs (100,000g ultracentrifugation) and soluble plasma (sup)5. results: hras-mediated cellular transformation (in ras-3 cells) triggers profound changes in the structure of nuclear chromatin, which is driven into the cytoplasm and released as cargo of evs. oncogenic dna is detectable in blood fractions of tumour bearing mice. while evs, ctdna and plts contain intermediate levels of mutant dna, rbcs contain only traces of this material. the highest hras copy number per ml of blood is found in wbcs (monocytes and neutrophils), which contain more cancer dna/cell than liver, spleen and bone marrow. depletion of neutrophils using anti-ly6g antibody results in an increase in ev-and ctdna-associated mutant dna in blood, suggesting the role of these cells in regulating the circulating levels of cancer cell-derived particles. uptake of dna-containing evs impacts the phenotype of myeloid cells, which adopt thrombo-inflammatory properties. these cells also retain cancer-specific transcripts and other cargo. finally, normal astrocytes treated with oncogenic evs also exhibit phenotypic changes and signs of genomic instability including formation of micronuclei. summary/conclusion: we propose that the process of leukocyte sequestration of circulating particles containing tumour-related nucleic acids renders these cells potentially usable as a novel liquid biopsy platform (leukobiopsy) in cancer. introduction: early diagnosis of colorectal cancer (crc) and precancerous adenoma patients is of vital importance. previously we profiled small extracellular vesicles (sevs) derived mirnas isolated from plasma, proposed a new promising biomarker category of crc patients. here we further gave a full landscape of circulating sevs derived rnas to explore and evaluate sevs based rna biomarkers for early detection of both crc and adenoma patients. methods: plasma sevs were isolated from 60 participants, including 31 early-stage crc patients, 19 adenoma patients, and 10 normal controls (nc), and characterized according to misv2018 guideline. the total sevs derived rna expression profile of all participants was investigated by next-generation sequencing (ngs). weighted gene coexpression network analysis (wgcna) was performed to categorize differentially expressed rnas, and t-distributed stochastic neighbour embedding (tsne) was adopted to distinguish crc, adenoma, from nc samples with the top-ranked genes in wgcna modules. rt-qpcr validation was performed in a cohort of 120 additional participants. results: a total of 1615 rna species (including mirnas, mrnas, and lncrnas) were found differentially expressed between plasma sevs in crc and nc participants. additionally, 888 rna species were differentially expressed between plasma sevs in adenoma and nc participants. 1160 rna species were differentially expressed between plasma sevs in crc and adenoma participants. wgcna categorized all rnas into 6 modules, which exhibited different expression trends during the carcinogenesis of crc. a 60-gene combined tsne model consists of the top 10 genes in each module could perfectly classify crc, adenoma, and nc samples. a 6-gene combined tsne model consists of the top 1 gene in each module could roughly distinguish crc and adenoma from nc, with only 1 sample misclassified. rt-qpcr assays also confirmed the potential classification ability of those genes in another validation cohort of 120 participants. introduction: although the concept of systematic "liquid biopsy" using bodily fluids is simple and elegant, the path of clinical reality has been challenging. recently, numerous tissue-specific biomarkers have been discovered in evs derived from blood, urine, cerebrospinal fluid, cell culture media, and a variety of other fluids. however, tracing the lineage of evs to their tissue of origin remains challenging due to their minute amount of cargo and unavailability of matching biopsied tissue and bodily fluids from the same patient. we recently demonstrated in three separate publications (dogra et. al; smith et. al; murillo et. al) , a new device (nanodld) for ev isolations, it's comparison with current technologies, bioengineered vesicles, and a detailed study of rna types present in small/ large vesicles, lipoproteins, and ago2 protein in different biofluids. in the present study, we aim to investigate the lineage of prostate derived evs in biofluids. methods: using our chip technology, we have isolated exosomes from prostate cancer cell lines and patient tissue, blood and urine samples. after exosome isolation, small rna libraries were prepared, and sequencing is carried out at icahn school of medicine and new york genome center using illumine sequencer hiseq2500. our nanofluidic pillar array is manufactured in an sio2 mask using optical contact lithography and deep ultraviolet lithography. results: our study revealed i) rna markers, which are exclusive to their prostate tissue of origin and are secreted in evs; ii) approximately 1-3% of prostate tissue-specific rna were discovered in evs; iii) over 77% (17 of 22 rna) of literature curated prostatespecific rna signatures were detectable in serum and urine evs from pca patients; iv) evs contained over 50-70% of noncoding rna (40-60% was mirna), while tissue predominantly yielded rrna (>60%); v) finally, gene set analyses generated that over 90% of evs rna were enriched for signalling pathways, yielding mirna-associated, non-canonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively monitor prostate tissue-specific biomarkers, identify tumour-specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. summary/conclusion: in summary, we have investigated patient matched tissue, serum, and urine derived evs in prostate cancer. we present a set of 68 prostatic rna in evs, which are enriched in noncanonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively track prostatic biomarkers, identify tumour specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. a multi-model, liquid biopsy approach for diagnosing and staging pancreatic adenocarcinoma introduction: pancreatic ductal adenocarcinoma (pdac) is the third largest contributor to cancerrelated death in the usa. since there is not yet a feasible technology to diagnose pdac early in the disease, 80% of patients are diagnosed at an advanced stage. moreover, for patients with confirmed pdac, standard imaging method has low sensitivity to detect early metastatic disease, which complicates the selection of therapy. to address these challenges, there has been great interest in developing minimally-invasive, extracellular vesicle (ev) based blood tests for pdac. to this end, we have integrated measurements of tumour derivedev rna cargo with circulating proteins and cell free dna (cfdna), and use machine learning algorithms to distill this multiplexed diagnostic to 1. diagnose pdac patients from healthy and disease controls and 2. distinguish pdac patients with distance sites of metastasis to guide their treatment. we make use of our lab's magnetic nanopore isolation technique to specifically enrich for tumour derived evs directly from patient plasma. methods: we have developed a high throughput nanofluidic sorting platform, which immunomagnetically isolates individual evs from plasma using magnetic nanostructures. however, our architectures is uniquely designed for massive parallelization allowing high throughput, robust processing of ml of plasma in minutes. we performed sequencing on a discovery set of patients and controls (n = 29). subsequently, we trained our panel of biomarkers using a training set of n = 47. finally, we validated the performance of our platform using an independent blinded test set of n = 145. results: the results of a blinded test set achieved an accuracy = 92% and an auc = 0.95 on binary classification of pdac patients versus those that were healthy or disease controls. in addition, we achieved an auc = 0.85 and accuracy = 89% with sensitivity of 90% and specificity of 88% on detecting occult metastasist. summary/conclusion: we developed a highly sensitive pancreatic cancer diagnostics by combining our nanomagnetic isolation platform for tumourderived ev isolation, rna sequencing, and machine learning. we isolated tumour-derived evs and profiled their rna cargo, combined with cfdna and ca19-9 for pancreatic cancer diagnosis. the predictive panels successfully distinguished non-cancer patients from pdac patients, and nodistant metastasis patients(m0) from distant metastasis patients(m1) for appropriate treatment. the resulting auc and accuracy from the independent blinded test set outperformed any individual biomarker, showing both the benefits and the robustness of combining multiple orthogonal biomarkers for pdac diagnosis. introduction: both hypertension and diabetes exhibit significant molecular changes to the vasculature that are associated with increased cardiovascular risk. here we examined the protein composition of large evs (l-evs) isolated from the plasma of hypertensive, diabetic and healthy mice to identify common and diseasespecific molecular changes. methods: we examined circulating l-evs isolated from transgenic mice expressing active human renin in the liver (ttrhren, a model of hypertension), ove26 type 1 diabetic mice, and their wild-type (wt) littermates. at 20 weeks of age mice were sacrificed and blood samples were obtained by cardiac puncture. l-evs were isolated from platelet-free plasma via differential centrifugation and protein content was assessed via mass spectrometry (ms). results: ttrhren mice exhibited increased blood pressure compared with ove26 mice or their wt littermates. (144.2 ± 7.6 vs 123.5 ± 4.9 [ove26] vs. 114.6 ± 5.7 mmhg [wt], p < 0.05). ms identified 297 independent proteins with at least 2 peptides per protein. of these, 163 proteins were found in all groups studied, 48 were exclusive to wt mice, 23 were exclusive to ove26 mice and 4 were exclusive to ttrhren mice. in addition, 22 proteins were observed with >1.5 fold change (fc) compared to wild-type mice, and 68 proteins were reduced by >50%. amongst the top ten differentially expressed proteins, fibrinogen was upregulated in both ove26 and ttrhren mice compared with wild-type controls. similarly trem-like transcript 1, sarcoplasmic/endoplasmic reticulum calcium atpase 3 and junction plakoglobin were all downregulated in both ove26 mice and ttrhren mice suggesting molecular changes common to both conditions. conversely, arginase was up-regulated in diabetic, but not hypertensive mice while carboxypeptidase was upregulated in hypertensive but not diabetic mice. summary/conclusion: taken together, these results show that the protein composition of circulating l-evs is altered in diabetes and hypertension and that both common and disease-specific changes may be detected. further analysis of these changes may lead to the identification of novel pathways associated with the pathogenesis of vascular injury in hypertension and diabetes. funding: this study was supported by grants (to db) from the canadian institutes of health research, an ontario early researcher award, and the canada foundation for innovation. understanding the role of endothelial cell-derived apoptotic bodies in inflammatory signalling and cell clearance in an atherosclerosis model of inflammation. introduction: apoptotic bodies (apobds) are a class of large (~1-5um) evs formed during apoptotic cell disassembly, that are becoming increasingly recognized as potential mediators of intercellular communication, e.g. via the transfer of proteins and other cargoes to target cells. during the inflammatory vascular disease atherosclerosis, endothelial cell (ec) apoptosis contributes to loss of barrier function and promotes the formation of plaques in regions of ec damage. although, experimentally, ecs generate an abundance of apobds, a specific role for ec-derived apobds (ec-apobds) in the progression of atherosclerosis remains poorly defined. methods: in the present study, a detailed in vitro characterization of ec disassembly was performed via flow cytometry, confocal live cell imaging and cytokine profiling, followed by function analyses of ec-apobds using a murine in vivo model of dead cell clearance. results: characterization of ec disassembly revealed that apobd formation in ecs is regulated by rho-associated, coiled-coil-containing protein kinase 1 (rock1), a process that can be pharmacologically inhibited using a rock-1 inhibitor, thereby providing tools for functional in vivo studies. the specific cargo and role in clearance of ec-apobds were then investigated. profiling of ec-apobds was performed via cytokine antibody array to reveal that ec-apobds generated under inflammatory conditions contain high levels of pro-inflammatory cytokines including mcp-1 and il-8, suggesting a potential role for ec-apobds in the propagation of inflammation during vascular disease. furthermore, the ability of ec-apobds to be cleared from the vasculature via phagocytosis was investigated, revealing that ec-apobds can travel to distal organs to undergo clearance. summary/conclusion: these findings provide important insights into the potential functions of ec-apobds generated under both non-inflammatory and inflammatory conditions and may contribute to future studies involving the therapeutic targeting of ec disassembly for the treatment of atherosclerosis. funding: this work was supported by grants from the national health & medical research council of australia (gnt1141732, gnt1140187) adipose mesenchymal stromal cell derived evs foster cardio-renal protection in the doca-salt hypertensive rat model introduction: cardio-renal syndromes (crs) are disorders of the heart and kidneys whereby "acute or chronic dysfunction in one organ may induce dysfunction of the other". stem cell-derived extracellular vesicles (evs) mediates the protection of the kidney from development of chronic kidney disease (ckd). we here investigated the potential of adipose-mesenchymal stromal cells derived evs (asc-evs) as therapeutic tools for the treatment of crs. methods: adult wistar rats were uninephrectomized and treated with a high-na+ diet and deoxycorticosterone-acetate (doca-salt) for 8-weeks (038/15; a02/16-61-15). evs were isolated by ultracentrifugation method. ev dimension, concentration and surface markers were characterized by nta, cytofluorimetric analysis and transmission electron microscopy. to characterize the role of evs in crs, doca-salt rats were injected weekly with asc-evs. systolic blood pressure was measured by the tail-cuff method. plasma creatinine and urinary protein excretion were determined by colorimetric assays and microalbuminuria by immune turbidimetric assay. qrt-pcr and western blot were conducted to evaluate fibrosis and inflammatory-related genes and proteins in the kidney and heart of doca-salt rats. immunohistochemistry was used to confirm matrix accumulation (a-sma) and immune infiltrate (cd68+ cells). results: multiple administration of asc-evs in doca-salt rats induced a protective effect on the kidney, by reducing tubular and vascular damage. kidney function was also conserved by ev treatment as detected by the normal glomerular filtration rate and the absence of proteinuria with respect to doca-salt untreated rats. ev administration significantly decreases the pro-inflammatory molecules mcp-1 and pai1 and reduce the recruitment of macrophages in the kidney. the mitigation of the inflammatory response by asc-ev infusion consequentially affected the development of fibrosis, as detected by the decrease in collagens (col1a1, col4a1) and fibronectin (fn) expression in respect to doca-salt animals. asc-evs were able to act in multiple organs, preventing fibrosis and inflammation also in the heart, therefore alleviating blood pressure rise during the 8-weeks of treatment in doca-salt rats. summary/conclusion: our results indicate that asc-ev administrations in hypertensive-induced ckd rats promote protection from renal damage, reduction of the inflammatory response and prevention of interstitial fibrosis in the kidney. asc-evs are also able to protect the cardiac tissue and to control blood pressure increase, displaying complex and multiorgan beneficial effects. introduction: alveolar macrophages (ams) tonically secrete extracellular vesicles (evs) containing suppressor of cytokine signalling 3 (socs3) protein. uptake of socs3-containing evs by alveolar epithelial cells is critical for restraint of cytokine-induced janus kinasesignal transducer and activator of transcription 3 (jak-stat3) signalling to promote homoeostasis in the distal lung. at steady state, ams exhibit suppressed glycolytic activity, a metabolic phenotype that promotes homoeostatic function. whether this glycolytic restraint is critical for am secretion of socs3 is unknown. in fact, to our knowledge, metabolic control over release of any ev cargo has never been explored in any cellular context. methods: immortalized mouse ams (mh-s) were treated with various doses of 2-deoxy-d-glucose (2-dg) and oligomycin, inhibitors of glycolysis and oxidative phosphorylation, respectively. primary rat ams collected by lung lavage were treated with an aqueous extract of cigarette smoke (cse) with or without 2-dg. metabolic activity was measured by seahorse assay, evs were quantified by nanoparticle tracking analysis, and vesicular (>100-kda) socs3 secretion was determined by western blot of conditioned medium. additionally, ams collected from wild-type (wt) and lsl-krasg12d mice bearing lung tumours 16 weeks after intrapulmonary ad-cre were cultured ex vivo in the presence or absence of 2-dg. vesicular (>100-kda) socs3 secretion was measured by elisa. results: in a dose-dependent manner, oligomycin inhibited, whereas 2-dg enhanced, socs3 and ev release by mh-s cells. treatment of rat ams with cse (1%) attenuated secretion of socs3, an effect that coincided with increases in glycolytic activity, and co-treatment of ams with 2-dg abrogated the inhibitory effect of cse on socs3 release. finally, ams collected from lsl-krasg12d mice exhibited a deficiency in socs3 secretion relative to wt ams, an effect that was reversible by overnight culture in the presence of 2-dg. summary/conclusion: in tandem, our data generated using in vitro and in vivo approaches demonstrate that am secretion of vesicular socs3 is down-regulated by glycolysis. we speculate that metabolic control over release of ev cargoes is a phenomenon of broad biologic relevance within and outside of the lung. introduction: bacterial extracellular vesicles (ev) are described to play roles in defence and resistance, pathogenesis and stress responses. cyanobacteria pioneered oxygenic photosynthesis, and are the ancestors of modern chloroplasts. we previously described that by deleting the gene encoding tolc (δtolc) in the model cyanobacterium synechocystis sp pcc6803 (s6803), a key player in protein-mediated secretion systems, a hyper-vesiculating phenotype could be obtained. the goal of this work was to understand why δtolc hyper-vesiculates. methods: isobaric tag for relative and absolute quantitation (itraq) was used for quantitative proteomic analyses of total cell extracts. ev were isolated as follows: cells were separated from the extracellular medium (em) by centrifugation (4400 g, 10 min) and filtration (0.2 µm pore-size filters). cell-free em was concentrated using centrifugal filters (mwco of 100 kda), and later ultracentrifuged for 3 h at 100000 g. the final ev fraction was suspended in growth medium. ev characterization was performed using tem, dls, nanosight, and by the detection and quantification of lps (lipopolysaccharides). detection of specific proteins in ev was carried out by western blot. copper (cu) levels were quantified by atomic absorption spectrometry (aas). results: a large-scale quantitative proteomic analysis was performed, resulting in the identification of several metal-related proteins with differential regulation in s6803 δtolc. both wild-type (wt) and δtolc cells were then challenged with different metals. compared to the wt, δtolc showed impaired growth only when exposed to cu, a co-factor for several proteins with roles in primary metabolism. the intracellular cu levels were quantified and δtolc accumulates threefold more cu than wt cells. we then asked whether the hyper-vesiculating phenotype observed could be linked to the stress induced by cu accumulation. in ev isolated from δtolc we detected the metallochaperone copm, a periplasmic cu-binding protein involved in cu-resistance mechanisms in s6803. in addition, cu could also be detected in isolated δtolc-ev. in addition, more ev were detected when s6803 wt cells were challenged with cu, in a cu-concentration dependent manner. summary/conclusion: these results support the idea that bacterial ev represent an alternative cu-secretion mechanism to deal with cu-induced stress. funding: fct phd grant sfrh/bd/130478/2017; feder-compete2020-poci-fct project: poci-01-0145-feder-029540. juan wang and maureen barr rutgers university, human genetics institute of nj, piscataway, usa introduction: extracellular vesicles (evs) function in intercellular communication. despite their physiological importance and biomedical relevance, knowledge of ev fundamental biology is not well understood, in part due to a lack of tractable animal systems. our analysis of environmentally-released c. elegans ciliary evs provides strong evidence that nematodes package cargo in evs that mediate inter-organismal communication, in analogy to intercellular signalling in mammals. we predict that conserved mechanisms underlie ev cargo sorting, biogenesis and signalling. cilia act as cell towers to both receive extracellular signals and to send information via ciliary evs. ciliary defects result in human ciliopathies including autosomal dominant polycystic kidney disease (adpkd). adpkd is a life-threatening disease that affects 1/800 and is caused by mutations in pkd1 and pkd2, which encode polycystin-1 and −2. in c. elegans and humans, the polycystins are architecturally similar, act in the same genetic pathway, function in a sensory capacity, localize to cilia, and are shed in evs, suggesting ancient conservation. moreover, ciliary ev biogenesis and shedding is an evolutionary conserved process from algae to worms to humans. by studying how cilia make and receive evs, we aim to uncover fundamental principles of how cells communicate using evs. methods: to study ciliary ev cargo sorting and biogenesis, we use genetically-encoded fluorescent-tagged ev cargo and superresolution zeiss airyscan confocal microscopy in living animals. results: we find that cargoes are sorted into distinct populations. in cilia, kinesin-2 motors and kinesin-3 klp-6/kif28 transport different ev cargoes to the ciliary tip and generate an ev cargo enrichment zone. from here, evs are shed and released into environment in a spatially and temporally regulated manner. ciliary ev biogenesis and release is regulated by mechanical pressure and ph. our work revealsat the single cell levelthat different evs are made in response to environmental stimuli, which may be important for ev signalling properties. summary/conclusion: cells exploit the spatiallyrestricted cilium and its sophisticated transport system to generate distinct populations of ciliary evs. how these ciliary ev communicate cellular messages awaits decoding. introduction: we recently demonstrated that recycling endosomes marked by rab11a generate exosome subtypes distinct in cargos and functions from late endosomes, which we collectively term rab11-exosomes. these exosomes are preferentially released from cancer cells in response to metabolic stress and promote adaptive changes in a xenograft model. here we use comparative ev proteomics in hct116 colorectal and hela cervical cancer cell lines to identify rab11-exosome signature proteins and screen for functional effects. methods: we analysed ev preparations by mass spectrometry using tandem mass tag® labelling to identify changes in ev protein cargo in response to glutamine depletion. candidate genes were subsequently knocked down in drosophila secondary cells, which permit visualisation of rab11-exosome biogenesis using fluorescence microscopy, and in human cancer cell lines. results: we show that accessory escrt-iii proteins, chmp5, chmp1 and ist1, are enriched on glutamine-depletion-induced evs and play a selective and conserved role in generating rab11-exosomes. they are, however, not required to traffic ubiquitinated cargos into late endosomes and lysosomes. escrt-0 components, thought to regulate trafficking of ubiquitinated cargos into intraluminal vesicles, are also required to make rab11-exosomes. in flies the escrt-0, hrs, localises to the limiting membrane of rab11-endosomes. comparative proteomics reveals other proteins enriched in rab11-exosomes, which also appear to be needed to mediate this novel exosome formation mechanism. summary/conclusion: we conclude that rab11-exosome subtypes are formed via a distinct mechanism requiring accessory escrt-iii components, suggesting a route to selectively target these exosomes. introduction: the tumour microenvironment consists of a complex network of host cells embedded within extracellular matrix. communication between these cellular compartments is critical for tumour progression and exosomes have emerged as important regulators of intercellular communication. while a number of studies have implicated exosomes in cancer progression, mechanisms controlling exosome transfer are not well understood. we developed three-dimensional (3d) culture models to evaluate the role of cues provided by the extracellular matrix in exosome release and uptake. methods: exosomes were isolated from cells in two-and three-dimensional culture via ultracentrifugation and characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. exosomes were labelled with fluorescent lipophilic dyes and uptake in recipient cells quantified by flow cytometry. results: cells cultured in 2d display decreased exosome release and increased uptake compared to 3d cultured cells. exosome release in 3d culture was inhibited with the exosome release inhibitors brefeldin a and gw4869, but was not significantly altered by knockout of rab27b. in addition, disruption of polarity signals provided by 3d culture did not impact exosome release or uptake in 3d, but induction of oncogenic hras increased both secretion and uptake of exosomes through activation of pi3 k signalling. summary/conclusion: release and uptake of exosomes is altered in 3d environments. these studies help provide insight into exosome production and uptake in vivo and have potential implications for therapeutically targeting exosome release and the development of exosome based therapeutic delivery vehicles. introduction: previous studies in our lab found that expression of r345 w-fibulin-3 induces rpe to undergo emt. the purpose of current study was to characterize the extracellular vesicles (evs) in rpe cells expressing wt-fibulin-3 versus rpe cells expressing r345 w-fibulin-3 and investigate the effects of these evs on rpe cell differentiation. methods: arpe-19 cells were infected with lentivirus with luciferase-tagged wild-type (wt)-fibulin-3 or luciferase-tagged r345 w-fibulin-3. evs were isolated from the media of arpe-19 cells by conventional ultracentrifugation or density gradient ultracentrifugation. transmission electron microscopy (tem) and cryogenic electron microscopy (cryo-em) were performed to study the morphology of the evs. the amount and size distribution of evs were analysed by nanosight tracking analysis (nta). ev protein concentrations were quantified using the dctm protein assay (bio-rad). ev cargo were analysed by unbiased proteomics using lc-ms/ms with subsequent pathway analysis (advaita). migration ability was evaluated in arpe-19 cells with or without the exposure of evs by conducting scratch assays. results: morphologically, tem imaging showed concave-appearing vesicles and cryo-em imaging showed spherical vesicles with two subpopulations of evs: a small group with diameters around 30 nm and a large group with diameters around 100 nm. moreover, tem and cryo-em showed an increased amount of small evs (~30 nm) in the mutant group compared to the wt group. this result was further confirmed by nta showing that, in the mutant group, the particle size distributions were smaller than the wt evs. no significant differences were shown in ev protein concentrations per particle between wt and mutant groups. our previous data suggest that the expression of r345 w-fibulin-3 causes rpe cells to undergo emt as evidenced by upregulated emt drivers and an increased migration ability. proteomic studies showed that evs derived from arpe-19 cells overexpressing wt-fibulin-3 contain critical members of sonic hedgehog signalling (shh) and ciliary tip components, whereas evs derived from rpe cells overexpressing r345 w-fibulin-3 contain emt mediators, indicating that ev cargo reflects the phenotypic status of their parental cells. ev transplant studies showed that exposing native rpe cells to mutant rpe cell-derived evs containing emt drivers, including tgf-β-induced protein (tgfbi), vim, and smad4, leads to an enhanced migration ability of rpe cells in a dosedependent manner. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms (2d static tissue culture flask vs 3d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in 2d t-flasks and 3d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both 2d and 3d culture systems. introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies methods: pca patients and age-matched healthy controls (hc) plasma (n = 6 in each group) were used to isolate sevs with 3 different isolation methods including commercial exoquick ultra kit, qev 35 and qev 70 size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd9 and cd81 commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev35 demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev70. furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods summary/conclusion: qev 70 method demonstrated better performance in isolating relatively pure sevs from human plasma; qev35 has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev35 but with the highest non-sev protein contaminations. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd9, cd63 and cd81, it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: 1) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and 2) the assessment of subpopulation target enrichment relative to the population average by leveraging tim4 as an unbiased, lipidbased ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasisassociated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr3 (her2+), t47d and mcf-7 (er+pr+), bt549 and mda-mb-231 (triple negative) breast cancer cell lines, as well as five mda-mb-231-derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her2 was broadly detected in her2+ skbr3 evs. interestingly, her2-t47d and mcf-7 evs also expressed her2 where it was highly enriched in its epcam+ subpopulations. itg α6, β3 and β4 were only found in triple negative and organotropic evs with itg β3 and β4 differentially enriched based on the organotropism. the population average of mda-mb-231 and lung-tropic evs had high expression of itg β4, where subpopulations of cd44+ evs showed positive enrichment while cd9+ and cd63+ evs showed negative enrichment. itg α5, β3 and β4 were absent in the bone-tropic cd81+ ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb-231 evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. op2.06 = pf17.06 heparan sulphate proteoglycans are required for ev-mediated delivery of multiple growth factors sara veiga, alex shephard, alex cocks, aled clayton and jason webber cardiff university, cardiff, uk introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du145 prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified 49 targets that bind to hs-gag chains, and also 108 different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of evintroduction: methamphetamine (ma) and related amphetamine compounds, which are potent psychostimulants, are among the most commonly used illicit drugs. neuroimaging studies have revealed that chronic ma abuse can indeed cause neurodegenerative changes in the brains of human ma abusers including prominent microglial activation throughout the brain. it is still unclear how chronic inflammation caused by ma abuse leads to long-term damage to the brain. with this in mind, we are particularly interested in studying the role of extracellular vesicles (evs) in eliciting chronic inflammation in ma exposed brains. in the present study, we focus on the role of a mirna, mir-29a-3p (mir-29a) in chronic ma exposure. here, we present novel data that shows for the first time how chronic ma impacts not only the biogenesis but also the ev associated mirna cargo thereby affecting the overall health of the neurons and glial cells in the brain. methods: -density gradient centrifugation for isolation of brain-derived vesicles -characterization of bdes by western blotting, nanoparticle tracking analysis and transmission electron microscopy -quantitative rt-pcr -digital droplet pcr -confocal imaging of dendritic spines and synapses results: in the present study, we show from both in vivo and in vitro studies that chronic methamphetamine (ma) treatment alters ev biogenesis and microrna (mirna) cargo. brain-derived evs (bde) isolated from frontal grey tissue of rhesus macaques that were administered ma in a chronic regimen revealed a significant increase in both number and size. further analysis revealed increase in biogenesis genes and increased levels of mirna, mir-29a-3p (mir-29a). in situ hybridization of the frontal brain area revealed that mir-29a was exclusively expressed in microglia and neurons. further, in vitro studies revealed that ev associated mir-29a elicited not only neuronal damage but also was able to activate microglia to release pro-inflammatory cytokines thereby inducing a chronic inflammatory cycle. finally, we show that an anti-inflammatory drug was able to rescue inflammation, mir-29a levels and synaptodendritic injury. summary/conclusion: in summary, our results present for the first time show that chronic ma exposure in the brain affects ev biogenesis and mirna expression. we further confirm that mir-29a can serve as potential marker to diagnose synaptic deficits for chronic ma addiction in humans. finally, we reveal that anti-inflammatory drug could rescue the ev biogenesis and reduces the secretion of mir-29a, thereby rescues synaptodendritic injury. our data further supports the use of the anti-inflammatory drugs as therapeutic interventions for ma addiction. funding: nida funding # r01da042379 blood-borne and brain-derived ectosomes/microparticles in morphineinduced anti-nociceptive tolerance deepa ruhela, veena bhopale, ming yang, kevin yu, eric weintraub, aaron greenblatt and stephen r. thom university of maryland school of medicine, baltimore, usa introduction: opioid pain treatment is impeded because chronic administration decreases analgesia, a condition called tolerance that prompts dose escalation contributing to morbidity and mortality. inflammatory interleukin (il)-1β is required for tolerance development, so we hypothesized that pro-inflammatory extracellular vesicles (evs) play a role. methods: evs with opioid administration were assayed in mice and humans. annexin v-positive, 0.1-1 µm diameter microparticles (mps) were assessed by flow cytometry in murine and human blood and in murine deep cervical lymph nodes that drain brain glymphatics. blood-borne exosomes (<100 nm) were assayed by tunable-resistance pulse sensing (trps). anti-nociceptive tolerance following morphine administration to mice was assessed by speed of tail removal from warm water. results: repetitive morphine dosing of mice to induce anti-nociceptive tolerance increased blood-borne mps by eightfold, and by tenfold in cervical lymph nodes. mps expressed proteins specific to neutrophils, microglia, astrocytes, neurons and oligodendrocytes. il-1β content of mps increased 68-fold. administration of an il-1β antagonist to mice diminished blood and glymphatic mps elevations and abrogated tolerance induction. intravenous polyethylene glycol telomer b that lyses mps and intraperitoneal methylnaltrexone that binds peripheral opioid-mu receptors and myeloid differentiation factor-2 to inhibit toll-like receptors, inhibited mps elevations and tolerance. neutropenic mice did not develop anti-nociceptive tolerance, elevations of blood-borne mps or cervical node mps expressing microglial proteins. elevations of blood-borne exosomes were not identified based on trps analysis. patients entering treatment for opioid use disorder exhibited similar mps elevations as do tolerant mice. summary/conclusion: neutrophil-derived mps containing il-1β are required for morphine-induced antinociceptive tolerance. funding: this project was supported by grant n00014-16-1-2868 from the office of naval research and an unrestricted grant from the national foundation of emergency medicine. evs are a conveyor of toxic dipeptide repeat proteins in c9orf72 als/ ftd models thomas jefferson university, philadelphia, usa introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterized by loss of motor neurons. in als, motor symptoms initiate focally and then progress gradually, distal from the initial focus. abnormal forms of als-associated proteins are physically exchanged between neuronal cells. pathogenic als proteins like sod1, fus and tdp43 are transmitted between cells by assisted mechanisms, mainly extracellular vesicles (evs), spreading toxicity and misfolding of native proteins within the recipient cells. an intronic g4c2 aberrant nucleotide repeat expansion in c9orf72 gene is the most common genetic cause of als. translation of this expanded region occurs by a process called repeat associated non-aug (ran) translation that produces five dipeptide repeats proteins (dprs), polyga, polygp, polygr, polypa and polyga. polyga, polygr and polypr are associated with toxicity in neurons. in this work we study the recruitment of these aberrant proteins into extracellular vesicles (evs) and the potential role of these evs in spreading toxicity between cells of the central nervous system. methods: to isolate the evs from cell culture media we isolated by ultracentrifugation the larger vesicles at 21,000xg and the smaller evs at 100,000xg. number, size and fluorescence of the vesicles were analysed by fluorescent nanotrack analysis (f-nta) and by cytoflex. the protein content of the vesicles was analysed by western blot (wb). to evaluate the potential toxicity of the evs, a transwell system (tw) was employed. neuron viability was assessed using live imaging techniques. results: nsc34 were transfected with reporter constructs expressing dprs tagged with gfp protein. by f-nta, cytoflex and wb analysis we assessed that all the five dprs were loaded in both the large and the small vesicles isolated from cell culture medium. by tw, nsc34 transfected with the dprs were put in contact with primary cortical neurons (cns) transfected with synapsin driven td-tomato for live imaging purposes. we observed that polygr+ nsc34 were able to cause a significant decrease in cns viability. we also observed that polygr+ evs associated toxicity was directly dependent on polygr length. this effect was reverted reducing the number of polygr+ evs treating nsc34 with gw4869. to understand the downstream effect of polygr+ evs in recipient cells we studied tdp43 mislocalization, ran-translation and activation of the integrated stress response, finding a dysregulation of all these potentially toxic pathways in neurons treated with polygr+ vesicles. summary/conclusion: concluding, dprs are actively secreted in evs and polygr+ vesicles cause the activation of toxic mechanisms in the recipient cells, possibly contributing to the spreading of als introduction: pregnancy is the a condition that profoundly mitigates symptoms of multiple sclerosis (ms) a complex disease characterized by immune dysfunction and neurodegeneration affecting 2.3 million people worldwide. serum exosomes, released by specific cells during pregnancy, modulate the immune and central nervous system function and contribute to pregnancy-associated suppression of experimental autoimmune encephalomyelitis (eae), an induced preclinical model of ms. extracellular vesicles (evs) are the new means for communication among cells. the aim of our study was to characterize the ability of human amniotic fluid stem cells-derived evs (hasc-evs) to antigen presenting cell function thus correcting immune dysfunction in eae. methods: amniotic fluids were obtained from human 16-17-week pregnant women. hasc-evs were collected by ultra-centrifugation. evs were characterized for their specific proteins, lipids and nucleic acids expression. the ability of evs to modulate immune responses was performed in vitro, testing the ability of evs to induce a tolerogenic phenotype in mouse bone marrow derived dendritic cells, and in vivo for their potential to suppress eae, induced by immunization c57/b6 female mice with mog35-55 peptide. results: we found that hasc-evs expressed high levels of galectin-1 and promoted a significant increase of the immunoregulatory enzyme indoleamine 2,3dioxygenase-1 enzyme in dcs. moreover in in vivo experiments administration of hasc-evs significantly reduced disease severity in eae. such effect was associated with reduced neurological deficits and suppression of pathogenic t helper 17 (th17) cells, and increased percentage of regulatory t cells (treg-foxp3+) cells. summary/conclusion: our findings unravel immunoregulatory effects of evs secreted by hascs. evs may represent a novel cell-free immune regulatory and regenerative therapeutic approach that can potentially mitigate immune dysfunction and promote remyelination. association of neuronal-derived extracellular vesicles cargo with cognitive decline in late middle life introduction: alzheimer's disease (ad) is characterized by a long preclinical stage during which phosphorylated tau pathology spreads in the brain leading to clinical symptoms. pathogenic tau spreads, in part, via extracellular vesicles (evs). we and others have demonstrated that tau cargoes of neuronal-derived evs (nevs) from blood can serve as biomarkers for ad. we aimed to examine whether nev tau cargo can predict cognitive decline in late middle age by leveraging samples from participants in the wisconsin registry for alzheimer's prevention (wrap) study. methods: we blindly immunoprecipitated nevs using antibody against neuronal l1 cell adhesion molecule (l1cam) from serum samples of 146 wrap participants who were cognitively unimpaired at baseline (mean age 62.4 ± 6.3 years old; 71.2% females; 42.5% apoe4 carriers), of whom half subsequently developed cognitive decline. we measured phosphorylated (p181 and p231) and total tau in nevs using electrochemiluminescence assays. we used linear regression models to identify differences between cognitive status groups including age, sex apoe status and the cognitive status*age interaction in the model. results: at baseline, we found trends for higher p181-(p = 0.07) and p231-tau (p = 0.05) levels in future decliners compared to stable participants. further, there were significant cognitive status*age interactions for ptau231 (p < 0.01), total tau (p < 0.001) and ptau181 (p < 0.05) with higher levels with increasing age in future decliners summary/conclusion: nev tau cargo differs between late middle-aged individuals at risk for ad with and without future cognitively decline even before decline occurs, presumably due to subclinical spread of tau pathology. further nev biomarker development may allow preclinical ad diagnosis. introduction: in the brain, circulating extracellular vesicles (evs) in the cerebrospinal fluid (csf) contain a variety of signalling factors, including proteins, enzymes, and rna transcripts. while evs have been implicated in many cell-to-cell signalling contexts, the vast majority of these studies are based on findings derived from cell culture conditions. thus, the ability to identify cell typespecific ev release from cellular subpopulations within the brain represents a critical barrier in the field. methods: to address this knowledge gap, we utilized a novel transgenic mouse model to determine the release of cell-type specific evs. here we report the exomap-2 mouse, which is designed to express an exosomal green fluorescent protein in response to expression of cre recombinase. specifically, the exomap-2 transgene was inserted at the mouse h11 locus and consists of (i) a broadly expressed cag promoter/enhancer, (ii) a floxed orf encoding mts-tdtomato, (iii) an orf encoding the exosomal protein acyltya fused to mneongreen (mng), and (iv) a 3ʹ utr containing the wpre element and polyadenylation signal from the bovine growth hormone gene. results: intracranial ventricular injections of the viral vector aav-ttr-cre, which drives cre recombinase expression from the choroid plexus-specific promotor of the transthyretin gene, leads to acyltya-mng expression in the choroid plexus. moreover, we observed that these mice released mneongreen-positive evs into the cerebrospinal fluid and also visualized the vesicles in the blood. furthermore, these mice displayed an accumulation of acyltya-mng fluorescence in the medial habenula. summary/conclusion: the results indicate that choroid plexus-derived evs are trafficked to the csf and the medial habenula, and more generally, that the exomap-2 mouse can be used to follow the trafficking of tissuespecific evs into biofluids and between tissues in vivo. introduction: large-scale colorectal cancer (crc) sequencing studies have shown that 93% of all tumours had at least one mutation in proteins implicated in the wnt signalling pathway. mutations in β-catenin have often been associated with the constitutive activation of wnt signalling pathway and has been established as a major driver of crc. one of the proposed mechanisms of activating wnt signalling involves extracellular vesicles (evs) as cellular couriers to transfer wnt ligands from one cell to another. however, the association of oncogenic mutant β-catenin with evs has not been studied. subpopulations of cancer cells with different mutational loads and behavioural variations lead to intra-tumour heterogeneity methods: integrative proteogenomic analysis showed the secretion of mutant β-catenin via evs. evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. silac-based quantitative proteomics analysis, immunofluorescence, biochemical analysis, qpcr and xenograft models were employed to unveiling the role of evs carrying mutant βcatenin. results: an integrative proteogenomic analysis identified the presence of mutated β-catenin in evs secreted by colorectal cancer (crc) cells. follow up experiments established that evs released from lim1215 crc cells stimulated wnt signalling pathway in the recipient cells with wild type β-catenin. silac-based quantitative proteomics analysis confirmed the transfer of mutant β-catenin to the nucleus of the recipient (rko crc) cells. in vivo tracking of dir labelled evs in mouse implanted with rko crc cells revealed its bio distribution, confirmed the activation of wnt signalling pathway in tumour cells and increased the tumour burden. introduction: there has been a significant increase in incidence of human papillomavirus 16 (hpv16) driven oropharyngeal cancer (opc) in developed countries. there is evidence that hpv alters the molecular cargo of exosomes released by opc. emerging evidence suggests that hpv integration within the human genome is associated with both genomic and transcriptomic alterations. consistent with previous studies, the genomic viral-cellular junctions were identified using dips-pcr method in 15 (88%) saliva samples collected from hpv16-driven opc. methods: morphology and molecular features of exosomes derived from three different saliva sampling methods: unstimulated saliva; acid-stimulated saliva; and salivary oral rinses were examined using transmission electron microscopy (tem), nanoparticle tracking (nta) and western blot analysis. hpv-16 dna detection in salivary exosome was determined by using qpcr method. proteome profile of salivary exosomes derived from both cancer-free controls and hpv16-driven opc patients was characterized using liquid chromatography-electrospray ionization-tandem mass spectrometry (lc-ms/ms). results: we demonstrate that unstimulated saliva had greater abundance of exosomes when compared to the other sampling methods. three common exosome markers (cd9, cd63 and cd81) were higher in unstimulated saliva. only salivary exosomes derived from hpv-driven opc patients had a detectable level of hpv-16 dna. the proteomic signature of salivary exosome was significantly (p < 0.01) different between cancer-free controls and hpv-driven opc. we found elevated protein abundance of five main glycolytic enzymes (i.e. phosphoglycerate kinase 1 (pgk1), glyceraldehye-3-phosphate dehydrogenase (gapdh), aldolase (aldoa) and lactate dehydrogenase a (ldha) in salivary exosomes derived from opc patients, suggesting a functional role of salivary exosome in the reciprocal interplay between hpv-driven opc and glucose metabolism. summary/conclusion: our data suggest that the development of a low-cost non-invasive saliva-based test using both salivary exosomal dna and protein may offer an opportunity to detect hpv-driven opc, that may be clinically useful in managing these patients. continuous in vivo release of mast cell derived extracellular vesicles from an implanted device spreads pro-inflammatory response in mice introduction: mast cells are important players of the immune system and they secrete a wide range of mediators during bacterial infections. mast cells are also able to release extracellular vesicles (evs). here, we report that mast cells communicate with each other in vivo by evs. methods: we isolated bone marrow-derived and peritoneal mast cells from gfp-transgenic and wild type mice. evs were separated from the conditioned media of these cells cultured in the presence or absence of lipopolysaccharide (lps). evs were characterised according to the misev2018 guidelines by flow cytometry, electron and fluorescent microscopy, trps, the spv lipid and the bca protein assays. separated ev-s were cultured with naïve mast cells, and tumour necrosis factor (tnf)-α production was tested by elisa and intracellular flow cytometry. gfp+ mast cells were seeded in diffusion chambers which were implanted into the peritoneal cavities of mice enabling us to investigate the continuous in vivo release of evs. uptake of gfp+ evs and tnf-α expression of peritoneal mast cells were tested by flow cytometry and fluorescent microscopy. results: here, we showed that bacterial lps-sensing mast cells release evs that in turn, induce tnf-α expression in resting mcs in vitro. moreover, we confirmed that evs are transmitted to other peritoneal mast cells in vivo spreading the pro-inflammatory response by inducing tnf-α secretion in peritoneal mast cells. summary/conclusion: ev communication between members of the mast cell network, play an important role in spreading and escalating pro-inflammatory responses to immune stimuli. our data may provide an explanation how the relatively rare tissue resident mast cells can play key roles in diseases such as autoimmune arthritis. the ability of small extracellular vesicles (sevs) to reprogramme cancer cells is known. integrins, receptors for extracellular matrix proteins, are major players in mediating sev functions. previously, we have reported that the αvβ3 integrin is detected in sevs of prostate adenocarcinoma (prca) cells and transferred into recipient cells in a paracrine fashion; however, its role and expression have never been explored in the most aggressive forms of prca, such as neuroendocrine prca (neprca). neprca does not express androgen receptor (ar) but does express neuron-specific proteins, such as aurora kinase a, synaptophysin and neuron specific enolase, that activate pro-tumorigenic pathways independently from the ar. methods: we isolated sevs from prca c4-2b cells using iodixanol density gradients and characterized them by immunoblotting and exoview. the experiments were performed in vivo by injecting subcutaneously, in nude mice, du145 cells treated with sevs expressing or lacking the αvβ3 integrin, and in vitro, by testing anchorage-independent growth of different cell lines treated with the same sevs. discarded human tissues from prca metastasis were analysed by immunohistochemistry (ihc). results: we demonstrate that a single treatment of prca cells with sevs significantly stimulates tumour growth and anchorage-independent growth. moreover, we show that one treatment with sevs, shed from c4-2b cells that express αvβ3, but not from the control cells that lack αvβ3, induces differentiation of prca cells towards a neuroendocrine phenotype and downregulates ar. finally, our ihc analysis shows coexpression of αvβ3 integrin and synaptophysin in neprca metastatic lesions. summary/conclusion: in conclusion, our current study shows, for the first time, that αvβ3 integrin expression in donor cells generates sevs that reprogramme recipient cells towards an aggressive tumour phenotype. funding: this study was supported by nci-p01-140043, r01-224769 to lrl. introduction: exosomes are small extracellular vesicles (sevs) that carry a variety of cargoes and have been shown to promote tumour cell motility and metastasis. cell motility is influenced by dynamic formation and stability of filopodia: actin-rich protrusions that extend from the leading edge and perform directional sensing. filopodia regulators such as fascin are upregulated in multiple epithelial cancers and can promote invasive phenotypes. however, how filopodia are induced and controlled by extracellular factors is poorly understood. here, we describe a role for sevs in regulating filopodia formation and tumour cell motility. we utilized b16f1 melanoma cells and ht1080 fibrosarcoma cells for fixed-and live-cell imaging to quantify filopodia numbers and dynamics in control and exosome-deplete conditions. itraq proteomics was used to identify sev protein cargoes that contribute to filopodia formation. in vivo experiments were performed using a chick embryo model for metastasis. results: inhibition of exosome secretion in cancer cell lines, via rab27a or hrs knockdown, led to decreased filopodia numbers. specificity to sevs was demonstrated by rescue experiments in which purified sevs but not large evs rescued the filopodia phenotypes of exosome-inhibited cells. live imaging of hrs-kd cells revealed that exosome secretion regulates formation and stability of filopodia. proteomics data and molecular validation experiments identified the tgf-beta coreceptor endoglin as a key sev cargo regulating filopodia formation, cancer cell motility, and metastasis. summary/conclusion: in this study, we identified exosomal endoglin as a regulator of filopodia formation and in vivo metastasis. these data are relevant to cancer as endoglin expression is altered in many cancers. in addition, endoglin is the disease gene for hereditary haemorrhagic telangiectasia, and may influence angiogenesis. overall, our data implicate sev-carried endoglin as a key cargo regulating filopodia. astrocyte-derived ev-mediated blood-brain barrier disruption shilpa buch, ke liao, susmita sil, fang niu and guoku hu university of nebraska medical center, omaha, usa introduction: the breach of the blood-brain barrier (bbb), resulting in ensuing neuroinflammation, is a key feature of hiv-associated neurological disorders (hands). while combination antiretroviral therapy (cart) has successfully suppressed peripheral viraemia, cytotoxicity associated with the presence of viral tat protein in tissues such as the brain, remains a significant concern. our previous study has demonstrated that hiv-1 tat can induce disruption of bbb by downregulation of tight junction (tj) proteins in human brain microvascular endothelial cells (hbmecs) and that this is regulated by the autophagic pathways. methods: evs were isolated from hiv tat-stimulated mouse/human primary astrocytes using the standard differential ultracentrifugation method and characterized by transmission electron microscopy, nanosight & western blot analyses. among the various mirs dysregulated in hiv tat -stimulated astrocyte ev cargo, mir-7 was found to be upregulated by realtime pcr. confocal microscopy identified uptake of astrocytic evs by hbmecs. functional assessment of astrocytic ev uptake by hbmecs involved cell permeability using transepithelial electrical resistance as well as trans-well endothelial cell monolayer permeability assays. results: hiv-1 protein tat-mediated induction of micrornas (mirs) in astrocyte-derived extracellular vesicles (adevs) regulated the permeability of bbb by targeting the expression of tj proteins in the hbmecs. exposure of hbmecs to tat-adevs resulted in down-regulation of the tight junction protein claudin 5, resulting in increased endothelial cell monolayer paracellular permeability. microarray data of tat-adevs demonstrated upregulation of several mirs compared to that of controls, among which upregulated mir-7 was identified to target the tj proteins using ingenuity pathways analysis. increased expression of mir-7 was validated in tat exposed astrocytes and tat-adevs. adevs loaded with mir-7 oligos showed similar effects as that observed with tat-adevs in inducing permeability in hbmecs. increased expression of mir-7 with downregulation of claudin-5 was also recapitulated in microvessels isolated from the brains of doxycycline-inducible hiv-1 tat transgenic mice (itat) mice and in lysates isolated from the frontal cortices of siv+ macaques/hiv+ autopsied brains. summary/conclusion: our findings demonstrated that tat-adevs containing mir-7 as an important mediator underlying tat-mediated disruption of the bbb. introduction: endogenous exosomes and related extracellular vesicles (evs) are potent nanoparticles released by all cells tested to date. the exploitation of their unique scaffolding for engineering next-generation drug delivery systems represents a major area of academic and commercial interest. the lag in exploiting this potential is in part due to our inability to measured extent and efficiency of modification, e.g., composition and drug loading. here we report a robust pipeline of optical tweezing combined with raman spectroscopy to molecularly characterize engineered evs and quantitatively assess extent of drug loading at single particle resolution. methods: evs derived from cell culture and isolated by ultracentrifugation were fused with synthetic liposomes to create engineered evs (eevs). these eevs were formed via well-established vesicle fusion techniques, namely (1) mechanical extrusion, (2) freeze-thawing, or (3) probe-tip sonication. prior to formation, calcein was encapsulated in the liposomes and used as a surrogate for soluble drug loading. laser trapping raman spectroscopy (ltrs) was used to optically trap single evs, before and after synthetic manipulation. raman spectral analysis was used to assess trapped eevs compared to pure standards to quantify ratiometric variation in chemical composition. results: raman laser trapping experiments confirmed that each formation method results in largely varying (1) extent of fusion between evs and synthetic calceinloaded liposomes, (2) efficiency of calcein loading, and (3) particle size. we could also quantify the molar amounts of liposome vs. ev molecules for single particles, revealing a great amount of variation from particle to particle. functional membrane proteins we left intact to varying degree across fusion methods. summary/conclusion: given the rising importance of analytical tools able to characterize extent of molecular loading for engineered evs, we believe this technology will be very useful, thus warrants further investigation for eev characterization across a variety of clinical applications. funding: randy carney, phd was supported by a research scholar grant, rsg-19-116-01-cdd, from the american cancer society. extracellular vesicles containing host restrictive factor ifitm3 inhibited zika virus infection of foetuses in pregnant mice through trans-placenta delivery allen z. wu nanjing university, nanjing, china (people's republic) introduction: zika virus (zikv) infection can lead to neurological complications and foetal defects, and has attracted global public health concerns. effective treatment for zikv infection remains elusive and a preventative vaccine is not available yet. therapeutics for foetus need to overcome blood brain barriers to reach placenta and require higher safety standard. methods: in the present study, we engineered mammalian extracellular vesicles (evs) to deliver a host restrictive factor, interferon-induced transmembrane protein 3 (ifitm3), for the treatment of zikv infection. results: our results demonstrated that the engineered ifitm3-containing evs (ifitm3-exos) were overall safe to the animals and suppressed zikv viraemia by 2 log10 s in the pregnant mice. moreover, the engineered evs effectively delivered ifitm3 protein across placental barrier and suppressed overall zikv viraemia in the foetuses to the basal level with significant reduction of viraemia in key foetal organs as measured by q-pcr. mechanistic study showed that ifitm3 was delivered to the endosomes/lysosomes where it inhibits viral entry to the host cells. summary/conclusion: our study demonstrates that exosomes can act as a cross placenta drug delivery vehicle to foetus and ifitm3, an endogenous restriction factor that is highly expressed in placenta, is a potential treatment for zikv infection during pregnancy. introduction: extracellular vesicles (ev) are natural and abundant nanoparticles capable of transferring complex molecules between neighbouring and distant cell types. translational research efforts have focused on co-opting this communication mechanism to deliver exogenous payloads to treat a variety of diseases. important strategies to maximize the therapeutic potential of evs include payload loading, functionalization of the ev surface with pharmacologically active proteins, and delivery to target cells of interest. methods: through comparative proteomic analysis (lc/ms) of purified evs, we identified several highly enriched and ev-specific proteins, including a transmembrane glycoprotein (ptgfrn) belonging to the immunoglobulin superfamily. leveraging ptgfrn as a scaffold for surface display, we generated evs with functional targeting ligands, including single domain antibodies (sdabs), single chain variable fragments (scfvs), single chain fabs (scfabs), and receptor ligands, on the surface to direct ev uptake to cell types of interest. biological activity of these engineered evs was assessed in an array of in vitro and in vivo assays and compared to untargeted controls. results: we engineered evs displaying anti-clec9a scfabs to target conventional type 1 dendritic cells (cdc1s), anti-cd3 scfabs to target t cells, and cd40 ligand to target b cells. in mice, systemic administration of anti-clec9a evs resulted in a 75% increase in the percentage of cdc1 cells that take up evs over controls. anti-cd3 evs resulted in both an increase in the percentage of ev positive t cells (3.75 and 3-fold for cd4+ and cd8+) and the number of evs per cell (15 and 7-fold for cd4+ and cd8+) in the blood. furthermore, in primary mouse dendritic cells, anti-clec9a evs loaded with sting agonist achieved a 15fold greater pathway induction compared to untargeted controls. preliminary in vivo data suggest that anti-clec9a evs reduce the required sting agonist dose 10-fold to achieve efficacy and induce anti-tumour responses, compared to control evs. summary/conclusion: these results demonstrate the potential of our ev engineering platform to generate novel ev therapeutics targeted to cell types of interest for pharmacologic payload delivery. a novel method for the delivery of cell-free therapy to foetuses with congenital anomalies: a proof of principle study lina antounians, louise montalva, gabriele raffler, maria sole gaffi and augusto zani the hospital for sick children, toronto, canada introduction: antenatal cell-based therapies are currently considered invasive for the foetus. a promising cell-free strategy that holds great regenerative potential for several organs is the administration of stem cell derived evs, whose cargo contains bioactive molecules that epigenetically regulate target cells. herein, we aimed to 1) assess the ability of evs to reach foetal organs when administered to the mother intravenously or intra-amniotically; 2) compare these administration routes on normal foetuses and foetuses with a congenital anomaly. methods: evs were isolated from rat amniotic fluid stem cell conditioned medium using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd63, hsp70, flo-1, and tsg101 (western). we injected rat dams with evs stained by exoglow™-vivo or saline (control) via maternal tail vein (iv) or intra-amniotically (ia) at e20.5. ia and iv injections were performed on dams carrying normal foetuses or foetuses exposed to nitrofen to induce congenital diaphragmatic hernia. after 24h, dams and pups were sacrificed. 3d high-sensitivity optical reconstructions of whole foetuses or micro-dissected foetal organs were imaged using the ivis® spectrum imaging system. ev fluorescence signal was compared between normal (n = 27) and nitrofen-exposed (n = 45) foetuses. results: both iv and ia injection routes were successful in delivering evs to foetal organs. no fluorescent signal was detected in saline only control. ia injections yielded higher signal than iv, and evs reached more organs with ia than iv injections. ia injected evs were detected in the lungs, gastrointestinal, and urinary tract of normal and nitrofen-exposed foetuses. nitrofen exposed foetuses had higher signal than normal foetuses. summary/conclusion: this proof of concept study shows that antenatal administration of stem cell evs is feasible with different routes. although maternally administered evs cross the placenta, ia injection is more effective at reaching foetal organs. further studies are underway to reproduce these findings in experimental models of various congenital anomalies. funding: cihr-sickkids foundation grant os22.5 introduction: safe, efficient and specific nano-delivery systems are essential to the current cosmetic, nutraceutical and therapeutic medicine sectors. the ability to optimise the bioavailability, stability, and targeted cellular uptake of bioactive molecules while mitigating toxicity, immunogenicity and off-target/side effects is of the utmost priority. ves4us is a european project, which aims to develop an innovative platform for the efficient production of extracellular vesicles (evs) from microalgae, which constitute a promising renewable bioresource (www.ves4us.eu). here we present characteristics of evs from several microalgal lineages, which offer the opportunity for a potentially developing a new and scalable tailor-made biogenic nanotechnology. methods: we cultivated a number of ev-producing microalgal species and developed protocols for ev isolation both at laboratory (differential ultracentrifugation) and pilot scales (tangential flow filtration). the physico-chemical characterization of microalgal evs was carried out according to the minimal information for studies of extracellular vesicles 2018 (misev-2018 guidelines): biochemical methods to verify the presence of specific ev-biomarkers, tuned for microalgal evs; dynamic light scattering (dls) and nanoparticle tracking analysis (nta) to assess the particles number and size distribution; electronic scanning microscopy (sem), atomic force microscopy (afm), and cryo transmission electron microscopy (cryo-tem) for imaging analyses; bilayer-specific fluorescence staining (f-nta) to test the purity of ev preparation. results: we identified microalgae as a novel natural source of evs that could constitute a cost-effective and sustainable way of mass-producing them. we screened 20 strains of microalgae and generated an "ev identity card" for each, which contained a variety of ev features relating to their biophysical, biochemical and biological characteristics in line with the misev-2018. our approach will next focus on the scalable production, surface functionalization and bio-engineering of selected microalgal evs. at the same time, their bioactivity will be explored using both in vitro and in vivo biological models. summary/conclusion: the ves4us consortium is investigating the potential of microalgae as novel ev bioresources. this research will attempt to bioengineer novel naturally-derived nanocarriers, microalgal evs, suitable for the development of future cosmetics, nutraceutical or therapeutic formulations. funding: this project has received funding from the european union's horizon 2020 research and innovation programme under grant agreement no 801338. sequence-specific rna trafficking to extracellular vesicles is conserved across cell types several sequences have been identified that act as a zipcode for preferential rna targeting into ev (evtropic) or for retention in parental cells (cell-tropic) . in this work, we aimed to compare the ev-tropic capacity of specific rna sequence motifs in promoting loading into ev, across different cell models representing the main cell types found in the body. methods: immune, epithelial and mesenchymal cell lines were transiently transfected with xenogeneic c. elegans micrornas (mirnas) containing ev-tropic or cell-tropic sequences and grown in culture. ev were isolated from the supernatant by differential (ultra)centrifugation. rna was extracted from both cell pellets and isolated ev fraction, and target mirnas were quantified by digital droplet pcr. distribution of cargo mirna across cells and ev was also analysed for chimeras of ev-and cell-tropic sequences. results: the mirnas containing an ev-tropic sequence were highly enriched on the ev fraction, with 1000-10,000 higher levels than in parental cells. contrarily, cell-tropic mirnas were only 10-100 times higher in ev. no significant differences were observed in the ev loading efficiency for the various ev-tropic motifs tested. mutations in the ev-sorting motif resulted in reduced ev loading. ev-tropic sequences consistently promoted mirna loading into ev across all the cell models evaluated, suggesting conserved biological mechanisms. summary/conclusion: we showed that rna loading into ev is dependent on the presence of defined evtropic rna motifs, and that sorting mechanisms are conserved across the major cell types tested. the highest loading efficiencies resulted in 0.001 mirna copies per particle on average, suggesting a limited scope for ev-tropic motifs for therapeutic rna loading into ev. funding: as, os and eli are fellows of the astrazeneca postdoc programme. introduction: coordinated activity between pancreatic islet cells is critical for the regulation of glucose homoeostasis. chronic exposure to diabetogenic factors such as pro-inflammatory cytokines, perturb islet cell crosstalk and β-cell function in diabetes. extracellular vesicles (evs) derived from cytokine-exposed β-cells modulate physiological and pathological responses to β-cell stress. however, the mechanisms governing this process remain largely unknown. we set out to test the hypothesis that β-cell failure in diabetes is mediated in part through β-cell autocrine release of pro-inflammatory evs which promote inflammation and inhibit βcell function. methods: pro-inflammatory cytokine-exposed evs (cytoevs) were generated using conditioned media from mouse min6 β-cell line treated with diabetogenic cytokines (tnfα, il-1β, ifnγ, 48h). evs were also isolated from human type 2 diabetic (t2dm) and lean non-diabetics (lnd) plasma. gw4869 (n-smase inhibitor) was used in the presence of cytokines to determine the effect of reduced ev concentrations on the restoration of β-cell function. proteomic and rna-seq analysis was conducted on min6 β-cell cytoev (vs. control ev) and cytoev treated mouse islets, respectively. results: assessment of ev concentrations from cytoev and human t2dm plasma revealed a~twofold increase (p < .05, vs. control (ctl) and lnd ev). immunofluorescence staining of cd9 and cd63 expression was significantly elevated in human t2dm pancreas (p < .05, vs. lnd). while acute inhibition of ev formation with gw4869 (5 µm) showed significant restoration in β-cell function (glucose stimulated insulin secretion assay, gsis) in cytokine-exposed mouse and human islets (~7 and 2 fold vs. cytokines alone, p < .05). moreover, functional assessment of mouse islets exposed to cytoev (48h) resulted in suppression of gsis (~55%, vs. untreated, p < .05). identification of cytoev content through proteomic analysis revealed a significant upregulation of the chemokine, cxcl10 (~40 fold vs. ctlev) and rna-seq analysis of cytoev treated mouse islets depicted a marked upregulation of transcripts associated with cxcl10-cxcr3 signalling (p < .001) and downstream pathways (e.g. nfκb; p = .016 and jak/stat; p = .021). furthermore, inhibition of cytoev (gw4869) with cytokines markedly decreased cxcl10 (~30%) and cxcr3 receptor (~65%) expression in min6 β-cells. summary/conclusion: these data suggests that cytokines elevate cxcl10 expression in β-cell ev to enhance inflammation-induced diabetes. this is mediated through ev-autocrine release of cxcl10 consequently activating cxcr3 signalling and downstream pathways to impair β-cell function in diabetes. synergy between 15-lipoxygenase and secreted pla2 promotes inflammation by formation of tlr4 agonists from extracellular vesicles introduction: damage associated molecular patterns (damps) are endogenous ligands that induce innate immune response, thus promoting sterile inflammation. during oxidative stress, stress-derived evs (stressevs) were found to activate toll-like receptor 4 (tlr4), but the activating ligands were not fully determined. additionally, several enzymes, among them 15-lipoxygenase (15-lo) and secreted phospholipase a2 (spla2) are induced during inflammation and were suggested to promote damp formation. methods: stressevs were produced from hek293 cells exposed to 10um a23187 and isolated with ultracentrifugation. 20:4 lysopi was oxidized for 10 min with 15-lo. additionally, synevs were prepared from phospholipids (pls), oxidized with 15-lo and hydrolysed with spla2. activity was measured by qpcr and elisa on wt and tlr4-ko macrophages. 15-lo oxidized 20:4 lysopi was analysed by mass spectrometry. spla2 activity was measured in synovial fluid from rheumatoid and gout patients using fluorometric assay. k/bxn serum transfer induced arthritis model on wt and tlr4 ko mice (c57bl/6 mice) with spla2-iia injection was used (approval no. u34401-14/2019/8 by mkgp of slovenia). results: stressevs released after oxidative stress were found to activate tlr4 with a gene profile different from bacterial lipopolysaccharide (lps). stressevs, 15-lo oxidized synevs, but only 15-lo oxidized lysopls activated cytokine expression through tlr4/md-2. hydroxy, hydroperoxy and keto products of 20:4 lysopi oxidation were determined by ms and they activated the same gene pattern as stressevs. furthermore, spla2 activity, which we detected in the synovial fluid from patients, promoted formation of tlr4 agonists after 15-lo oxidation. injection of spla2-iia into mice promoted k/bxn serum induced arthritis in tlr4-dependent manner. summary/conclusion: both 15-lo and spla2 are induced during inflammation, therefore these results imply the role of oxidized lysopls in stressevs in promoting sterile inflammation through tlr4 signalling. the formation of tlr4 agonists is enzyme driven so it provides an opportunity for therapy without compromising innate immunity against pathogens. funding: h2020-msca-itn project tollerant (grant no. 642157), slovenian research agency (project no. j3-9257 to mmk, research core no. p4-0176 to rj). monocytes traffic extracellular vesicles to damaged muscle and adopt a novel immunophenotype to support muscle regeneration russell g. rogers, akbarshakh akhmerov, weixin liu, lizbeth sanchez and eduardo marbán smidt heart institute, cedars-sinai medical center, los angeles, usa introduction: extracellular vesicles (evs) are secreted membrane vesicles that carry bioactive molecules such as mirnas, mrnas, proteins, and lipids to modify recipient cell behaviour. we recently demonstrated evs secreted by cardiosphere-derived cells (cdc-evs) augment endogenous muscle regeneration in mdx mice, a model of duchenne muscular dystrophy, when delivered intravenously. in parallel, macrophages preferentially accumulate surrounding small regenerating myofibers in cdc-ev treated mdx muscle. however, it is currently unclear how intravenous cdc-evs home to dystrophic muscle and exert their therapeutic bioactivity. methods: fluorescently-labelled and unlabelled cdc-evs were infused into the contralateral femoral vein of wild-type mice with unilateral muscle injury induced by bacl2. injured and uninjured muscles were dissected 24h following infusion and subjected to optical imaging, immunohistochemistry, and confocal microscopy. this experiment was repeated using clodronate liposomes to deplete endogenous monocytes/macrophages. next, rna-seq was preformed on bone marrow-derived m1, m2, and cdc-ev (mcdc-ev) polarized macrophages from mdx mice. conditioned media (cm) from these macrophages were tested in an in vitro model of myogenesis. lastly, small rna-seq was performed on evs secreted by m1, m2, and mcdc-ev macrophages. results: when delivered intravenously, cdc-evs naturally home to injured, but not uninjured, skeletal muscle. cdc-evs were detected in the interstitium adjacent to non-muscle cells, macrophages, and within surviving myofibers. after depletion of monocytes/ macrophages by clodronate liposomes, the presence of cdc-evs in the injured muscle was attenuated. bioinformatic analyses indicate cdc-evs confer a novel immunophenotype to mdx macrophages with features of both m1 and m2. indeed, mcdc-ev cm promotes myoblast proliferation and supports myogenic differentiation. interestingly, mcdc-ev evs have a unique mirna signature and contain several mirnas with known roles in myogenesis. summary/conclusion: these data indicate circulating monocytes traffic cdc-evs to damaged muscle where they adopt a novel immunophenotype to support muscle regeneration. we propose mcdc-ev macrophages mediate their pleiotropic effects via paracrine factors, possibly including evs. introduction: microglia, the immunocompetent cells of the cns, play an important role in maintaining cellular homoeostasis in the cns. these cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. the contribution of microglial-derived extracellular vesicles (m-evs) to the maintenance of cns homoeostasis is unclear. in addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to m-evs is scarce. methods: here, we analysed the effects of m-evs produced in vitro by either tnfα-activated or non-stimulated microglia bv2 cells. we showed that m-evs are internalized by both mouse bv2 and human c20 microglia and that the uptake of m-evs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. consistently, exposure of microglia to m-evs increased the protein expression of the autophagy marker, lc3b-ii, and promoted autophagic flux in live cells. to elucidate the biological activities occurring at the transcriptional level in c20 microglia exposed to m-evs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted rna sequencing. results: inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia exposed to m-evs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. summary/conclusion: we demonstrate that in vitro produced microglial evs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homoeostasis. funding: this work was financed by hasselt university and by efro through the interreg v grensregio vlaanderen nederland project trans tech diagnostics. evaluation of plasma extracellular vesicles as biomarkers for longevity xin zhang a and virginia kraus b a laboratory medicine center, nanfang hospital, southern medical university, guangzhou, guangdong, 510515, p. r. china, guangzhou, china (people's republic); b division of rheumatology, duke molecular physiology institute, duke university school of medicine, durham, usa introduction: extracellular vesicles (evs) have emerged as key indicators and effectors of ageing. although plasma concentrations of evs decline with age, the ev biomarkers associated with ageing and longevity are not fully understood. recently, our group found an age-related decline of plasma evs associated with immune cells during normal human ageing. our study aims to evaluate the association of plasma evs with longevity. methods: plasma samples were selected from the established populations for epidemiologic studies of the elderly study subjects (n = 48): half dying within 2 years (short-lived group) and half surviving ≥10 years (long-lived group) after the blood draw; all matched for age (median age 77.3 ± 1.7 years, range 72-80), gender (50% female), and race (50% white/50% black). the samples were acquired under donor consent and irb approval of duke university. evs were separated from the plasma samples, and profiled based on the surface markers of haematopoietic stem cells (hscs), mesenchymal stem cells, immune cells, skeletal muscles, cardiac muscles and adipocytes (cd81, cd9, cd29, cd63, cd8, cd4, cd68, cd14, cd56, cd15, cd19, cd235a, cd41a, cd34, cd31, hla-abc, hla-g, hla-drdpdq, cd90, cd73, cd105, m cadherin, ryr1, ryr2, fabp4, dlk1). the percentages of evs expressing each tested molecule were determined using a high-resolution multicolour bd lsr fortessa x-20 flow cytometer as we recently reported. graphpad prism 8.0 software was used for statistical analysis. results: we found significantly increased percentages of cd9+, hla-abc+, cd31+ and cd41a+ large evs (1000-6000 nm) in the long-lived compared to the short-lived group. none of the tested surface marker expressing medium (100-1000 nm) or small (<100 nm) evs showed differential percentages between the shortand long-lived groups. summary/conclusion: evs carry surface markers from their parent cells. cd9 is expressed by hscs and immune cells. cd9 regulates homing of human cord blood cd34+ hscs, and delivers a potent cd28independent costimulatory signal to activate t cells. hla-abc, the key human immunogen, is expressed by nucleated cells and platelets. cd31 is expressed by hscs, immune cells and epithelial cells, and cd31 + plasma evs declined with age in healthy people. cd41a is expressed by hscs, megakaryocytes and platelets, and is functionally relevant for hsc maintenance and haematopoietic homoeostasis. our preliminary data suggest that hscs and immune cell associated plasma evs (cd9+, hla-abc+, cd31+, cd41a+ large evs) inform on health status related to longevity. introduction: it is anticipated that stem/progenitor cells-derived extracellular vesicles (spc-evs) will rapidly progress towards clinical studies, and the development of reproducible, efficient, scalable and costeffective process for their production is expected to boost the therapeutic applications of evs-based products. in addition, the use of defined serum-/xenogeneic(xeno)-free culture medium formulations could result in substantial improvements for spc-evs production in terms of reproducibility, stability and quality, while ensuring the approval of regulatory agencies. the main goal of this work is to develop a full-controlled manufacturing platform for the spc-evs production. methods: human mesenchymal stromal cells (msc) were expanded in a xeno-free microcarrier-based bioreactor culture system operating in fed-batch feeding mode and after 10 days the conditioned medium was collected. different methods for spc-ev isolation/purification from the msc-derived conditioned medium, including chromatography were compared and the the quality of the final product obtained was characterized by different methods according to misev, including nanoparticle tracking analysis, lipidomics and western blot. moreover fourier-transform infrared (ftir) spectroscopy was evaluated in terms of its implementation as a standard technique for the identification and characterization of evs. results: after 10 days of msc expansion under dynamic conditions, we collected 1.3 l of conditioned medium with approximately 0.5 million evs/msc. a combination of a pretreatment with a nuclease for the digestion of dna/chromatin with a purification using strong anion exchange chromatography led to the best results so far in terms of evs isolation. of notice, by ftir spectroscopy, it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture conditions tested. summary/conclusion: the platform established herein could be applied to the production of wellcharacterized spc-evs targeting their biomedical use in different settings (e.g. as drug delivery systems), as well as evs from other parental cells lines (i.e. dendritic cells) in therapeutic settings as cancer. ultrasensitive protein detection for quantification of extracellular vesicles in human biofluids enables comparison of isolation techniques dmitry ter-ovanesyan, maia norman, wendy trieu, roey lazarovits, george church and david walt wyss institute, boston, usa introduction: extracellular vesicles (evs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. one of the main challenges in studying evs and using them in diagnostics is a lack of suitable methods to quantify evs that are sensitive enough and can differentiate evs from similarly sized lipoproteins and protein aggregates. we propose using ultrasensitive single molecule array (simoa) assays to quantify evs by immunoisolating and detecting ev transmembrane proteins in microwell arrays. we developed single molecule array (simoa) assays using the quanterix hd-x analyser for the quantification of evs using the tetraspanins cd9, cd63, and cd81. simoa allows for the detection of single proteins using arrays of femtoliter wells, turning elisa into a digital immunoassay. we then used these assays, together with an additional assay for albumin, to compare commonly used ev isolation methods from plasma and cerebrospinal fluid (csf): ultracentrifugation, precipitation (exoquick), and size exclusion chromatography (sec) using the izon qev columns. we further used these assays to rapidly optimize and improve sec by comparing different sec resins and column dimensions in both plasma and csf. results: in comparing our simoa assays to traditional elisa with the same antibodies, we found that the simoa assays were more than 100 times more sensitive, detecting the tetraspanins in samples where the proteins were undetectable by elisa. given the high dynamic range and high-throughput capabilities of simoa, we were able to comprehensively compare relative ev yields and ev purity for different isolation methods of evs from plasma and csf. we provide average tetraspanin and albumin levels to directly compare the methods. we also tested different sec resins and provide data for custom sec columns that outperform izon qev and allow for fine tuning of different ratios of evs to albumin. summary/conclusion: our results highlight the utility of quantifying evs using ultrasensitive simoa assays for tetraspanins. we were able to rapidly simoa to rapidly evaluate different ev isolation methods in csf and plasma. in general, the experimental framework we present could be easily applied to evaluate new ev isolation methods, or applied to any other biological fluid. thus, we think simoa is a powerful new tool for relative ev quantitation. introduction: the protein profile of extracellular vesicle (ev) subpopulations has been shown to contain valuable disease information, notably in cancer. currently, techniques aiming to find ev proteins that associate together mainly focus on transmembrane proteins, while methods that also probe cytosolic proteins generally resort to a combination of affinity capture, elution, and lysis, which limits throughput. to allow the high-throughput analysis of both membrane and cytosolic ev proteins, we optimized a total extracellular vesicle antibody microarray (tevam) incorporating fixation and heat-induced epitope retrieval (hier), then leveraged it to perform combinatorial protein profiling of evs from colorectal cancer (crc) cell lines ht29 and sw403. methods: arrays of iggs targeting surface protein markers were incubated overnight with evs purified from cancer cell line supernatants. hier optimization was carried out through variation of buffer contents, presence or absence of prior permeabilization, as well as incubation time and temperature, for a total of 38 conditions. a431 evs, previously profiled with other methods, were used as a model during the optimization. cytosolic protein hsp90 and membrane marker egfr, both with high expression in a431 evs, were probed and the results used to compare hier conditions. following hier treatment, protein targets were detected through incubation with primary antibodies and fluorescent secondary antibodies or streptavidin. the resulting optimized tevam workflow was used to phenotype ht29 and sw403 evs through probing of trios of surface (2) and internal (1) protein targets. results: the selected tevam protocol successfully maximized hsp90 signal while minimally affecting egfr detection, enabling simultaneous analysis of surface and internal proteins. profiles of more than 450 combinations, featuring integrins, claudins, cytokines, and other key actors of cancer-relevant pathways, were obtained for ht29 and sw403 evs, revealing coexpression patterns that highlight the biomolecular heterogeneity both within and between crc cell line evs. summary/conclusion: using tevam, intra-and extravesicular proteins can be detected simultaneously in evs immobilized based on surface protein content, yielding extensive combinatorial protein profiles with significance for health and biomarker research. characterization of evs using orthogonal techniques identifies discrete ev populations from a mouse dendritic cell line bryce killingsworth a , timothy traynor b , joshua a. welsh c , aleksandra dakic a , jason savage a , kevin camphausen d , kenneth aldape a and jennifer jones a a laboratory of pathology, national cancer institute, national institutes of health, bethesda, usa; b laboratory of pathology, national cancer institute, national institutes of health, gaithersburg, usa; c laboratory of pathology, national cancer institute, national institute of health, bethesda, usa; d radiation oncology branch, national cancer institute, national institutes of health, bethesda, usa introduction: extracellular vesicles (evs) have the potential to serve as valuable biomarkers for patient response to cancer therapy. however, development of robust ev-based clinical assays relies on knowledge of ev concentration and diameter distribution. many different methods exist to measure the size and concentration of evs, and each method exhibits strengths and limitations. it is important to use orthogonal methods for determination of these important properties of ev preparations. here, we use dendritic cellderived evs to demonstrate that some ev analysis methods can give a biased interpretation of both diameter and concentration. through comparison, we highlight why orthogonal assays are essential in providing measurement reliability. methods: dc2.4 mouse dendritic cells were cultured in flasks containing a total of 1.2 l of ev-depleted media (10% fbs, centrifuged 18 hr. x 100,000 g.) when cells reached 80% confluency, conditioned media was collected, depleted of debris with two 10 min. x 2,500 g spins, and concentrated down to~5 ml using a pall jumbosep 100 kda mwco filter. the ev concentrate was purified from protein using an izon qev-10 column, with 5 ml fractions collected. the protein content of the ev-containing fractions was analysed by a280, pierce bca, and bioanalyzer. the diameter distribution of the evs was determined by nanoparticle tracking analysis (nta), resistive pulse sensing (rps), flow cytometry (fcm), and electron microscopy (em.) concentration was compared using nta, rps, and fcm. evs were further analysed by protein mass spectrometry and rna sequencing. results: we have identified two distinct populations of evs with our dc2.4 preparation, one highly abundant population with a power-law distribution, whose peak diameter is below 60 nm, and a second, less abundant population with a peak diameter at approximately 140 nm. these two distinct populations and their relative concentration were not detectable with all analysis techniques. based on cross-platform measurements, these populations appear to have distinct compositions that warrant further investigation. summary/conclusion: the use of orthogonal methods allowed the detection of two discrete populations of evs which was not possible on some platforms and would have resulted in a biased perspective of the sample composition. this work has highlighted the need for orthogonal measurements to be conducted by pairing techniques that do not have the same biases. introduction: extracellular vesicles (evs) are nanosized vesicles shed by all cells that serve vital roles in cell-to-cell communication. tumour-associated ev subpopulations vary in molecular content (lipids, proteins, nucleic acids, small molecules), enabling minimally invasive spectroscopic analysis for a wide variety of cancers. here, we use surface-enhanced raman spectroscopy (sers) in combination with a novel plasmonic substrate for global chemical composition analysis of cancerous and non-cancerous populations of evs to determine distinguishing surface characteristics. methods: evs were isolated from ovarian cancer (ovca) patient serum samples by differential ultracentrifugation. a new hybrid nanoplasmonic scaffold comprised of a microscale biosilicate diatoms embedded with silver nanoparticles (agnps) was used for sers measurements. the substrate was incubated with cysteamine to positively-charge the agnps (responsible for the sers enhancement) so that evs could attach (evs are naturally anionic). in a typical experiment, 40 μl of~108 particles/ml evs per sample were incubated with the porous substrate surface, which was inverted on a glass cover slip for raman interrogation. principle component analysis (pca) was used to compare the spectra and determine distinguishing characteristics between populations from tumour and non-tumour sources. we also trypsinized evs before sers analysis to see the extent of influence the surface molecules play in localizing the evs to the agnp "hot spots." results: a total of 8 clinical samples (7 ovca and 1 non-malignant control) were tested in combination with ovca skov-3 cell line evs. simple pca was able to separate clinical samples according to disease subtype and major peaks were identified to provide chemical content analysis. each sample exhibited inherent heterogeneity but clustered together in a distinguishable way from the others. summary/conclusion: despite innate heterogeneity within single samples (i.e., evs isolated from a single patient sample), evs isolated from clinical samples could be easily distinguished from each other using our hybrid sers substrate, with minimal sample processing, a label-free approach, and only a few microlitres of sample. our study using this novel plasmonic material demonstrates its potential for use as a component in next-generation diagnostic platforms. introduction: single-particle analysis is critical for understanding extracellular vesicle (ev) heterogeneity. yet such techniques remain technically challenging due to low detection sensitivity and presence of variable amounts of "contaminants," including lipoproteins. the high degree of structural similarity between evs and lipoproteins in size, density, and chemical composition, results in their co-isolation using any of the standard ev isolation techniques. here we introduce laser trapping raman spectroscopy (ltrs) as a wellsuited, label-free, and non-destructive tool to distinguish evs from various lipoprotein species at single particle resolution. methods: ev samples were isolated from skov-3 cell culture supernatant by differential ultracentrifugation and their raman spectra measured. as the most abundant lipoproteins in ev isolations from human biofluids are sub-micron low density lipoprotein (ldl), very low density lipoprotein (vldl), and high density lipoprotein (hdl) particles, these were purchased as pure components and also measured by ltrs. ldl and vldl were then spiked-in to isolated evs to mimic "contaminated" post-isolation ev samples. raman spectra were analysed by principal component analysis (pca) using a custom matlab script. results: ldl and vldl have been observed to adhere to ev surfaces in vitro after standard isolation techniques. we could readily distinguish pure vldl, ldl, and hdl standards according to their raman spectra. pca revealed distinction of skov-3 evs from both ldl and vldl. pca also differentiated skov-3 evs incubated with ldl from skov-3 evs incubated with vldl. extent of ldl and vldl adherence to evs could be observed and quantified. summary/conclusion: through raman and pca, classes of lipoprotein and evs can be identified and quantified when co-incubated. ltrs is a quantitative single-ev analysis technique that can be used to differentiate between lipoprotein classes and evs when incubated together. this technique allows for analysis of evs where standard isolation methods fall short. introduction: extracellular vesicles (evs) are endogenous membrane-derived vesicles that shuttle lipids, proteins or nucleic acids between glia and neurons, thereby promoting neuronal survival and plasticity in the cns and contributing to neurodegenerative conditions. although evs hold great potential as cns theranostic nanocarriers, the specific molecular factors that regulate neuronal ev uptake and release are currently unknown. methods: we used a combination of patch-clamp electrophysiology and ph-sensitive dye imaging to examine stimulus-evoked ev release in individual neurons in real time. results: whereas spontaneous electrical activity and the application of a high-frequency stimulus (hfs) induced a slow and prolonged fusion of multivesicular bodies (mvbs) with the plasma membrane (pm) in a subset of cells, the neurotrophic factor bfgf (basic fibroblast growth factor) greatly increased the rate of stimulusevoked mvb-pm fusion events and, consequently, the abundance of evs in the culture medium. proteomic analysis of neuronal evs demonstrated bfgf to increase the abundance of the v-snare vesicle-associated membrane protein 3 (vamp3, cellubrevin) on evs. conversely, knocking-down vamp3 in cultured neurons attenuated the effect of bfgf on ev release. introduction: heat shock proteins (hsps) function as chaperones under both normal and pathologic conditions. as chaperones they assist in protein folding, in holding protein complexes for current or future activation, and in degradation of senescent proteins for recycling of components and display for immune surveillance. during stressful situations, hsp quantities and/or activities increase as cells and tissues seek protection from insults. these insults can result in the cell surface display of hsps, which can then lead to the surface display of hsps on extracellular vesicles (evs). hsps present on the cell surface or in the extracellular space are regarded as "danger signals" in an ancient biologic paradigm. hsp-accessorized evs may act as "danger boli", carrying not only the hsps, but hundreds of components of the stressed parental cell, capable of prompting differential responses depending on the status of the recipient cell. methods: clarified/filtered plasma from patients suffering from neurologic maladies (cancer, brain injury, multiple sclerosis) was incubated with peptides designed to bind hsps. the evs congeal under these conditions and are pelleted (microfuge) and washed with increasing-stringency buffers. we lysed the evs and subjected them to metabolomic analyses (focused on lipids) or assayed them on phosphokinase arrays. results: we show that evs from the blood of patients suffering from brain tumours, or from tbi, or from ms, possess distinct metabolomes compared to blood evs from healthy donors. we found hundreds of differentially-expressed lipids amongst the patients vs the healthy donors. the levels of annotation and identification for these compounds ranges from level 4 (low, no matches in databases) to level 2 (high, annotation matches to known database components). in addition, we found differences in phosphorylated kinases as cargo in these evs between patients with matched primary vs recurrent gliomas, and among tbi/stroke patients compared to healthy donors. summary/conclusion: hsp-accessorized evs present different metabolomic and phosphokinase content which may serve as biomarkers in a "liquid biopsy" setting, but may also play roles in the pathobiology of neurologic diseases. introduction: methamphetamine (ma) has deleterious effects to both peripheral organs and the central nervous system. the rewarding properties and addictive potential of ma are correlated with increased synaptic dopamine availability following alterations in dopamine and vesicular monoamine transporter function. in rodents, ma alters brain mirna expression and the mirna content of serum extracellular vesicles (ev). here we examined plasma evs isolated from human subjects actively using ma (ma-act) for size, concentration, protein markers, and mirna content. methods: plasma samples from 10 ma-act, and 10 controls (ctl) were obtained from the methamphetamine abuse research center. plasma evs were evaluated by vesicle flow cytometry (vfc) for size, concentration, and surface protein markers. vfc antibodies included markers for a pool of tetraspanins (cd9, cd63, and cd81), platelet evs (cd41), pro-coagulant evs (annv), and red blood cell evs (cd235). next plasma ev isolated by size exclusion chromatography were analysed by qpcr on taqman® array human microrna a + b card set v3.0. fold change was calculated by δδcq between ma-act and ctl for mirna expressed in ≥ 60% of samples in at least 1 group. we identified the top 20% of ranked mirna by fstatistic; of these, the mirna of interest for ma-act were identified by at least a (i) 1.2 fold change in expression, (ii) area under the receiver operating characteristic curve of 0.75, and (iii) glass's δ of 1. for mirna of interest correlations to additional ma variables were conducted, along with ingenuity pathway analysis of predicted gene targets. tobacco use was controlled for. results: vfc data show that the size (~110 nm) and concentration (~7.5 x 1010 particles/ml) of all plasma evs is comparable between ma-act and ctl groups. in addition, the plasma evs primarily consist of tetraspa-nin+, annv+, or cd41+ evs, and to a much lesser extent cd235+ evs. of the 207 mirna expressed in ma-act and/or ctl plasma evs, there were 110 mirna that have at least a 1.2 fold increase or decrease in ma-act. 10 mirna were identified to be of interest in ma-act based on fold change, effect size and diagnostic potential, compared to ctl. further, 5 of the 10 mirna correlate with a ma associated variable, including frequency of use and age of first use. together the 10 mirna best cluster subjects based on ma-act status, not tobacco use. finally, the predicted gene targets of the 10 mirna are associated with canonical pathways linked to ma. summary/conclusion: ev mirna expression in ma-act subjects was unique to ctl participants, suggesting that ma may affect ev communication among cells. the differential mirna expression also implicates a role for evs in behavioural and physiological effects specific to ma, and suggests that there may be changes in expression of mirna that are relevant to specific drugs of addiction, as well as to a spectrum of drug-mediated addiction disorders. bone marrow-derived extracellular vesicles may alter the ageing phenotype of murine haematopoietic stem cells. sicheng wen a , jill kreiling b , mark dooner c , elaine papa c , michael del tatto c , yan cheng c , mandy pereira c , peter quesenberry c and laura r. goldberg d in natural ageing of haematopoietic stem cells (hscs) is unclear. we tested the hypothesis that bone marrowderived evs (bm-evs) can modulate the ageing hsc phenotype. methods: we flushed bone marrow from old (24-26month old) and young (6-8-week old) c57/bl6 mice and collected bm-evs by differential centrifugation (2000 × g for 30 min, supernatant collected and centrifuged 100,000 × g for 1 hour, bm-ev pellet collected and quantified by nanoparticle tracking analysis). we injected old mice with 2 x 10^9 young bm-evs via tail vein, daily x 3 days. control mice were injected with age-matched bm-evs or vehicle alone. we euthanized the mice one month post-injection, harvested whole bone marrow (wbm) and highly purified hscs (lineage negative/c-kit+/sca-1+/cd150+; lsk-slam) and tested stem cell function in competitive bone marrow transplants (4-5 recipients/group). results: at 6 months post-transplant, wbm from old mice exposed to young bm-evs exhibited a statistically significant decrease in engraftment when compared to wbm exposed to age-matched bm-evs (percent average donor chimerism ± sem: 15% ± 5% (young evs) vs. 61% ± 14% (old evs)). for lsk-slam from old mice, we observed a trend towards decreased engraftment when exposed to young bm-evs and a trend towards increased engraftment potential when exposed to old bm-evs (percent average donor chimerism ± sem: 7% ± 4% (young evs), 27% ± 10% (old evs), 15% ± 2% (vehicle)). these findings are consistent with our previous data showing that, in contrast to highly purified hscs which develop impaired stem cell function with ageing, total un-separated old wbm actually has increased engraftment capacity when compared to young wbm. of note, we found that the classic myeloid skewing by old lsk-slam was partially reversed by exposure to both young and old bm-evs. finally, consistent with the known increase in highly purified hscs with age, our preliminary data showed that old mice exposed to young bm-evs had an approximately 7-fold decrease in the number of lsk-slam cells in marrow, indicating that bm-evs may influence agerelated changes in hsc number. summary/conclusion: these preliminary data suggest bm-evs may play a role in modulating hsc ageing phenotypes, potentially altering engraftment capacity, lineage fate, and lsk-slam population size. future studies delineating the molecular mechanisms underlying these ev-mediated effects could provide key insights into normal haematopoietic ageing. funding: this work was supported by the nih grants 5p20gm119943, dk112808-01a1 and by the savit foundation. oral with poster introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr. results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom20. mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the biogenesis of evs is neutral sphingomyelinase 2 (nsmase2), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase2 is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase2 inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles. methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo [1,2-b] pyridazin-8-yl) pyrrolidin-3-yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated 5xfad mice with 10 mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay. results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, 5xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting 20% of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human post-mortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of 20 mdd subjects and 20 hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed. results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd9. tem images showed the typical cup-shaped morphology with sizes mostly between 30 and 200 nm. preliminary sequencing results revealed that mir-33a-5p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir-33a-5p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. op3.04 = ps15.04 combining nanomagnetic isolation and artificial intelligence to guide the treatment of traumatic brain injury zijian yang a , kryshawna beard b , david meaney b , danielle sandsmark b , ramon diaz-arrastia a and david issadore c a university of pennsylvania, philadelphia, usa; b university of pennsylvania, philadelphia, usa; c department of bioengineering, university of pennsylvania, philadelphia, usa introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of 7 protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores (600 nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the 7 protein biomarkers (tau, uchl-1, nfl, gfap, il6, il10, and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl1 were elevated in mtbi patients compared to controls (p < 0.05), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il6 and tnf-with tau from glur2+ evs showed 88% accuracy with 80% sensitivity and 100% specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. l1cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l1cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l1cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l1cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l1cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd9, cd63 and cd81) as well as l1cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l1cam appears. we also immunocapture l1cam from csf and plasma and perform western blots for the internal and external domains of l1cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l1cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l1cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l1cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l1cam in plasma is likely not coming from the brain. this data call into question the utility of l1cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant 3d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed 3d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in 6-month-old wt mice, (n = 6/groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for 15% of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and non-amplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed 968 proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy5y cells with n-myc amplified sk-n-be2 cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be2 cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. op3.08 = ps15.08 introduction: quantification and characterization of single extracellular vesicles (sevs) based on surface markers can aid in dissecting the heterogeneous landscape of ev subpopulations. we and others have demonstrated the potential of imaging flow cytometry (ifc) to perform sev characterization. we recently showed release of protoporphyrin (ppix) positive sevs by 5-aminolevulinic acid (5-ala) dosed glioma cells, in vitro and in vivo. rickfels et al. also used ifc to demonstrate the enrichment of cd63+/cd81+ evs in the plasma of glioma patients. herein, we performed in vitro studies to characterize ev subfractions using 5-ala as well as ev and cns specific surface markers. methods: we use ifc to characterize evs released by glioma using 5-ala, fluorescently labelled ev (cfda-se, cd81) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd81) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that 58% are tenascin c positive, 2.7% are egfrviii positive and 1.6% are 5-ala positive. there was only a minor overlap (<16%) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs (2.5-fold), tumour specific evs (2.8-fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: f. graminearum (fgr) and f. oxysporum f. sp. vasinfectum (fov) are severe fungal pathogens of cereals and cotton, respectively. fgr and fov cause economic losses and threaten food and fibre supplies worldwide. understanding host-pathogen interactions is crucial for developing new strategies for disease control. we are determining whether extracellular vesicles (evs) have a role in the interaction between fungal pathogens and their host plant. methods: we isolated evs from fgr and fov by sizeexclusion chromatography and characterized them by nta and tem. evs from fgr and fov are between 100-300 nm and have morphology similar to evs reported for other fungi. we performed label-free quantitative proteomics to describe the protein cargo of evs from fgr and fov, including a comparative study of evs from fov grown on different media: czapek dox (cd) and saboraud's dextrose broth (sdb). results: a total of 658 proteins were detected in fgr evs and, according to prediction software effectorp, 12.5% of these were potential effectors. similarly, 70% of ev proteins do not contain signal peptide indicating that packaging into evs is a novel mechanism of secretion for these proteins. notable fgr ev proteins include lipases, proteases and synthases for toxins and chitin. fov produced evs in similar quantities in both growth media tested, but ev protein cargo differed between them. there was a 39% overlap in proteins identified in the 465 cd and the 658 sbd ev proteins. in general, ev proteins were involved in metabolism, cell wall architecture and oxidoreduction, with 15.4% and 12.9% of potential effectors, respectively. polyketide and toxin synthases, proteases and effectors were present in both types of fov evs. summary/conclusion: this new fungal ev isolation method was rapid, yielded high-quality evs, and did not submit particles to high centrifugal forces. our data revealed that both fgr and fov produce evs enriched with proteins that could alter host immune responses or facilitate fungal infection. furthermore, the protein composition of fov evs was dependant on culture conditions. this supports a potential role for fungal evs in disease progression in plants and provides the foundations to pursue the role of evs in plant-fungal interactions with the potential to identify new targets for disease control. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll 2 receptor (tlr2) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il-12 and il-6, which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells. objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow fieldflow fractionation (af4) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp-1) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk-2 epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with 50 to 50 nm (ev1) and another with 100 to 120 nm (ev2). ev1 induced more tnf-alpha, il-6, ip-10 and ccl20 than ev2. it was also more effective in promoting t. cruzi infection in epithelial cells. summary/conclusion: t. cruzi released two ev populations that affects differently host cells. identification of these evs composition might help to better understand the role of evs in the modulation of t. cruzi infection funding: fapesp, cnpq and capes op3.11 = ps15.11 commensal bacterial extracellular vesicles act as carriers for norovirus sutonuka bhar, melissa jones, annalise galbraith and mariola edelmann university of florida, gainesville, usa introduction: human norovirus (hunov) are one of the most common causes of gastroenteritis and, along with inducing morbidity and mortality by diarrhoea, have a massive economic impact resulting in approximately 60 usd billion each year in healthcare costs and missed worker productivity. development of anti-viral therapies for hunov has been hampered by the lack of robust in vitro cultivation systems. several cell types support viral replication but only produce modest amounts of virus due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays. noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr. isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and 0.2um filtration. co-purification of norovirus with the evs was checked. ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid50 assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems. detection of bacterial extracellular vesicles in blood from healthy volunteers kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of 11.5 µg/ml of plasma, as geometric means ≥11.5 µg/ml were nearly equal. hevs were detected at 48 µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f200x electron microscope and measured between 40-90 nm, and hevs were between 60-160 nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: new zealand (nz) has a population of just 4.8 million people with a remote geographical location in the pacific ocean. its unique culture, food-based industries and ethnic population make nz an invaluable place for extracellular vesicle research into all areas. however, as for many places in the world, standardization of methodologies, training and access to appropriate equipment is challenging. methods: the hub for extracellular vesicle investigations (hevi) is a virtual research centre established in 2017 with three-year seed funding from a university of auckland strategic research initiatives fund. two staff members are employed to support training, education and optimization of methods. the hevi is guided by a governance group providing valuable input from australasian experts in evs. results: since 2017 the hevi has organized 2 research symposia, 2 hands-on training days, hosted 2 international students as well as providing one-on-one training for 41 individuals from universities and institutes across nz. training is provided on multiple isolation and characterization methods and tailored to individuals access to essential equipment without bias towards individual manufacturers or techniques. travel funding has supported 38 people to attend conferences and workshops for the purposes of education, networking and research dissemination. the hevi also provides support for project design with 10 grants awarded to hevi members and a number of manuscripts in submission for publication. the embo practical course "extracellular vesicles: from biology to biomedical applications" is organized each year by a group of 4 researchers active in the ev field in collaboration with the embl advanced training center in heidelberg. the course focuses on training phd students and postdoctoral researchers who enter or are already active in the field of ev research. given the large number of methods and protocols that is being used by researchers in the ev field, the organizers aim to provide practical guidance to new researchers and teach them appropriate skills. methods: participants obtain theoretical knowledge and hands-on experience on different ev purification and characterization techniques, such as fluorescent labelling, density gradient centrifugation, size exclusion chromatography, electron microscopy, flow cytometry and nanoparticle tracking analysis and on databases like ev-track and funrich. in addition, the organizers and invited lecturers from several different research areas explain which strategies are used to understand the role of ev in biomedical applications and give an overview of the current state of the field of ev research. results: the course therefore covers a broad range of topics important in the ev field, including heterogeneity in ev subpopulations, mechanisms of ev cargo selection, ev biogenesis, pre-analytical variables, therapeutic and diagnostic use of ev, and in vivo functions of ev. group discussions are facilitated and stimulated via assignments to analyse data obtained during the practicals and to critically evaluate literature. participants also have the opportunity to present their own research during poster presentations and ask for feedback from organizers and invited lecturers. summary/conclusion: among the participants selected for the course, a large geographical distribution is reached, including researchers from the newer eu member states and from outside of europe, to ensure a broad geographical distribution of the knowledge gained during this course. introduction: on 25 october 2019, we organized the 1st lugano exoday, first initiative in the southern switzerland to bring together resident researchers and european experts in the field of extracellular vesicles (evs). the workshop, centred on "technical challenges of extracellular vesicle research" aimed to highlight technical requirements and advances in the evs area, focusing on isolation, characterization and tracking. methods: the workshop started with a lecture by dr. cecilia lässer, from the university of gothenburg. the rest of the workshop was divided in two working groups (wg), each introduced by a keynote lecture followed by presentations by young researchers and a round-table discussion. wg1, introduced by dr. mercedes tkach, from the institute curie in paris, focused on recent advances on evs characterization and isolation. wg2 was centred on evs tracking and introduced by dr. frédérik verweij, from the institute of psychiatry and neuroscience of paris. results: dr. lässer opened the workshop with a comprehensive review and introduced recent developments in the evs field. the first wg discussed different isolation methods, focusing on ultracentrifugation, size exclusion chromatography and immunoprecipitation-based techniques. supported by the keynote speakers, the participants agreed that the best approach to optimize the isolation process consists in the combination of different techniques. wg2 shared insights about new strategies to visualize and tracking evs, focusing on how to improve the routinely approaches used, defining optimal criteria for evs labelling and imaging. all the participants had an in-depth overview on the requirements and the state-of-the-art techniques currently in use for the isolation, characterization and tracking of evs. summary/conclusion: the transferable knowledge acquired during the workshop ensures participants to remain up-to-date with the advances in the field of evs. as our ultimate goal is to create a competence centre in southern switzerland around the field of evs, the workshop was an invaluable opportunity to intensify collaborations between resident laboratories and broaden the scientific exchange with laboratories of the experts hosted during the event. given the success of this first workshop we are already working to prepare the second edition and make the event a recurring appointment. funding: supported by the swiss national science foundation the role of core facilities and emerging technologies in maximizing rigour and reproducibility of ev quantification and characterization and following misev guidelines rachel derita a and andrew hoffman b a thomas jefferson university, philadelphia, usa; b university of pennsylvania school of veterinary medicine, philadelphia, usa introduction: it remains very clear in the field of extracellular vesicle (ev) research that the rapid rate of increase in publications and expansion of interdisciplinary clinical ev interest has created the need for increased standardization and access to the appropriate technologies to uphold these standards. as the first core facility in the usa with the sole intention of creating a space where users can both isolate and characterize evs, we provide a central location for the facilitation of ev research via access to multiple technologies (both established and emerging) such as resistive pulse sensing, nanoparticle tracking analysis, ultracentrifugation, high-performance liquid chromatography, flow cytometric analysis of evs and additional immune or fluorescence-based ev characteri zation techniques. methods: we surveyed a group of leading scientific investigators and researchers in varying stages of their scientific careers in the mid-atlantic region of the us. the survey data demonstrate applications of greatest current and future interest to be employed in a shared lab resource. results: the current demand is highest for isolation services, ultracentrifugation and nta, with a gradually increasing demand for immunophenotying analyses such as the exoview chip array, fluorescent nta and flow cytometry. we additionally present strategies and data-based examples of how shared resource facilities can facilitate multifactorial and rigorous ev characterization in accordance with misev guidelines, and encourage collaboration among ev researchers. summary/conclusion: in order to answer the larger remaining questions in the ev field such as the isolation of specific ev subsets, ev tracking between cells and the use of evs for biomarker discovery and drug delivery, it is essential that shared resource facilities interact not only with investigators, but with each other to integrate the necessary resources to progress. programme to assess the rigour and reproducibility of extracellular vesicle-derived analytes for cancer detection national cancer institute, rockville, usa introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par20-053, is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par16-267/ 277, successfully funded 7 r01 and 4 r21 grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, 2018); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, 2019). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, 2019). summary/conclusion: drs. sudhir srivastava and matthew young are the program directors for the par which began accepting applications on 5 january 2020. this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute introduction: early detection of cancer as well as monitoring cancer treatment are important to improve cancer care. diagnostics for cancer are mainly based on tissue biopsies and re-biopsy during treatment is challenging. moreover, current diagnostics are expensive, time-consuming and have low-throughput. therefore, liquid biopsies are expected to bring the next breakthrough in cancer diagnostics. in liquid biopsies tumour-secreted material is isolated from body fluids and subsequent analyses thereof allow for non-invasive diagnostics. one type of tumour-secreted materials are extracellular vesicles (evs), which are schedded from tumour cells. evs are surrounded by a lipid bilayer, whichs composition resembles the plasma membrane of their parental cell. as many tumours are driven by over-expression or upregulation of transmembrane proteins e.g. growth factor receptors, detection of the later on evs holds promise for early tumour detection and treatment monitoring. methods: for the immuno-pcr evs were first affinitycaptured on magnetic beads, allowing immobilization of purified evs as well as evs secreted into cell culture medium or spiked into plasma. afterwards each sample was divided and affibody-dna-conjugates directed against different targets were added. affibodies are small affinity proteins, which often are developed as high affinity binders for tumour imaging, making them suitable probes in the presented assay. after washing, the bead-ev-affibody-dna-complexes were analysed for the immobilized dna-amount via qpcr. results: via the presented immuno-pcr evs secreted from the non-small cell lung cancer cell line h1975 as well as the ovarian cancer cell line skov3 were analysed. the immuno-pcr method allowed the detection of the tumour-associated membrane receptors epidermal growth factor receptor (egfr), receptor tyrosineprotein kinase erbb2/her2 and insulin-like growth factor 1 receptor (igf1r). different levels of membrane receptors depending on the ev source and concentration were detected. summary/conclusion: the presented immuno-pcr showed to be a comparably fast and robust method for detection of tumour-associated membrane receptors on evs derived from cancer cell lines with medium through-put and is currently further developed into a method for liquid biopsy for non-small cell lung cancer patients. introduction: introduction. evs produced by cells can originate from different cellular compartments and evs in complex biofluids may originate from many different cell types. traditional biochemical analysis, which reports on the total composition of all evs in a sample can't adequately resolve this heterogeneity. single vesicle analysis methods can, if they have the necessary specificity, sensitivity and speed. flow cytometry (fc) is capable of rapid and quantitative analysis of individual particles, but conventional fc-based assays lack the specificity and sensitivity to measure individual evs. assays that combine sensitive instruments with ev-selective sample staining can measure individual evs with accuracy and precision. to better understand the nature and origins of ev diversity, we used single vesicle fc (vfc) to quantitatively measure vesicle number, size, and surface cargo expression on individual evs. methods: methods. evs in culture supernatants (293 t, a431, u87, thp-1, sh-sy5y) were used neat or enriched by standard methods including differential centrifugation or ultrafiltration. evs from platelets (plt) and red blood cells (rbc) were induced by ionophore treatment of washed cells, and measured in diluted supernatant. evs were stained with a membrane-selective dye and fluorescence-labelled antibodies using a commercial vfc assay kit (cellarcus biosciences), measured using a commercial flow cytometer (cytoflexs, beckman coulter), and data analysed using fcs express v6 (de novo software). vesicle size, fluorescence intensity, and antibody binding were calibrated using appropriate vesicle and beadbased standards and essential controls performed as recommended by the miflowcyt-ev reporting guidelines. results: results. to assess the compositional heterogeneity of evs, we first characterized the expression of tetraspanins (tss; cd9, cd63, cd81, cd82, cd151, cd53, cd231) on evs released from cultured cell line and primary cell cultures. we find quantitative differences in the expression of ts on evs from different cell types that generally reflected the expression on the cell of origin, with most ev types expressing detectable amounts at least one of the common ts molecules (cd9, cd63 or cd81) but generally not all three. in evs from some cell types, ts expression was uniform across the ev population (cd9 on evs), but evs from other cell types differentially expressed tss, with some evs expressing no detectable ts (rbc evs). intracellular cargo labelled genetically using fluorescent proteins (egfp or mneongreen) or fluorogenic enzyme substrates (cfse) were measured in individual evs and revealed distinctive associations between ev surface and internal cargo. summary/conclusion: conclusions. high resolution measurement of cargo on/in individual evs can help interpret ev heterogeneity in terms of cell of origin, signals carried, and effects on target cells. integrated omics reveal conserved and divergent modulation of cardiovascular disease by tissue-entrapped extracellular vesicles introduction: fewer than 50% of patients develop both vascular and valvular calcification, implying differential pathogenesis. while circulating extracellular vesicles (evs) act as biomarkers of cardiovascular diseases, tissue-entrapped evs are implicated in early mineralization but their contents and function are unstudied. we developed an innovative method to isolate and study evs from fibrocalcific tissue and investigated entrapped ev cargoes in human cardiovascular diseases. methods: human carotid artery endarterectomies and stenotic aortic valves were obtained from 27 donors under irb-approved informed consent. tissues underwent enzymatic digestion, ultracentrifugation, and a 15-fraction optiprep density gradient. global proteomics was performed on intact tissue, each optiprep fraction, and ev-enriched pooled fractions; the latter also underwent mirna-seq. fractionated samples were also studied by cd63 immunogold electron microscopy (tem) and nanoparticle tracking analysis (nta). high confidence mir targets were predicted by targetscan, pathway analyses utilized the biocarta/kegg/reactome databases, and protein-protein interaction networks were built in string. results: vesicle-associated pathways were increased 5.1x (p < 0.01; 15/30 vesicle-related top go terms) in proteins common to intact arteries and valves (n = 1,411). proteomics found 24 ev markers to be highly enriched in the four least-dense optiprep fractions of arteries and valves, while extracellular matrix and mitochondria were predominant in denser fractions, as confirmed by tem/nta. proteomics and mirna-seq of tissue evs quantified 1,104 proteins and 123 mir cargoes linked to 5,182 target genes. pathway networks of proteins and mir targets common to artery and valve tissue evs revealed a shared regulation of rho gtpase and mapk intracellular signalling cascades. 179 proteins and 5 mirs were significantly altered between artery and valve evs (q < 0.05); multi-omics integration found that evs differentially modulated cellular contraction and p53mediated transcriptional regulation in vascular and valvular tissue. summary/conclusion: our findings delineate a novel strategy for studying tissue-entrapped ev protein and mir cargoes and identify critical roles that tissue-resident evs play in mediating cardiovascular disease. funding: this study was supported by a research grant from kowa company (ma) and nih grants r01 hl136431, r01 hl141917 and r01 hl147095 (ea). mir-20a regulates exogenous cd82 expression on proliferation, invasion, migration and angiogenesis of gastric cancer zhengzhou university, zhengzhou, china (people's republic) introduction: to investigate the possible mechanism of mir-20a regulating the expression of exosome cd82 on proliferation, invasion, migration and angiogenesis of gastric cancer, and to study the application value of cd82 in the early diagnosis and prognosis of gastric cancer. methods: the gastric cancer cell line mgc-803 was used as the research object. the exosomes were extracted from the culture supernatant of mgc-803 by exosome extraction kit. the extracted exosomes were identified by transmission electron microscopy and western blotting. the expression of cd82 in exosomes was detected by elisa. the expression of cd82 in exosomes and cd82 in whole blood and serum were detected by western blot. they were randomly divided into blank group (mock) and mir-20a lentivirus experimental group (mir-20a group). the lentivirus control group (mir-20a/con) was transfected into cells. qrt-pcr was used to verify the status of mir-20a after transfection; western-blot was used to detect the expression of cd82 and downstream erk1/2, akt and m tor proteins; mtt assay, cell colony formation assay, transwell migration assay for cell proliferation, invasion, and migration. a nude mouse xenograft model was constructed to observe the growth of transplanted tumours,microvessel density (mvd) was detected by immunofluorescence, and distant metastasis was recorded. results: the expression of cd82 in exosomes was detected by elisa and western blot. the expressions of cd82, akt, erk1/2 and m tor in mir-20a group were significantly lower than those in mir-20a/con and mock groups. cd82 protein is positively correlated with downstream protein levels.the growth rate and cell invasion ability of mir-20a group were significantly lower than those of mir-20a/con group and mock group. the weight of the nude mice in the mock group and the mir-20a/con group decreased, while the weight loss in the mir-20a group was not significant. the tumours in the mir-20a/con group and the mock group showed invasive growth accompanied by abundant microvessels, while the mir-20a group had smaller tumour volume and uniform cell distribution. only a small amount of microangiogenesis was observed, and no obvious necrotic area was observed. summary/conclusion: mir-20a affects the proliferation, invasion, migration and angiogenesis of gastric cancer mediated by akt/erk/m tor signalling pathway by regulating the expression of exosome cd82. streamlined detection and quantification of plasma extracellular vesicles and their protein cargo by high-performance nanoscale flow cytometry and label-free mass spectrometry introduction: nanoscale flow cytometry (fc) and mass spectrometry (ms) are useful for profiling ev surface proteins and performing bulk ev proteomics, respectively. this study sought to develop pre-analytical and analytical pipelines for ev protein profiling that are applicable to clinical studies. methods: to optimize plasma ev detection and quantification by fc, modifications of instrument settings and serial dilutions of platelet-free plasma (pfp) and antibodies were tested for improved separation of signal from noise and reduction of event coincidence and swarming. the high-performance flow cytometry (hpfc) platform was used to assess the effect of time (1, 2, 3, 5, 9, 24, 48 or 120hrs) between blood draw (into acd, nacit, edta or heparin) and blood processing, on ex-vivo release of evs from blood cells. label-free ms was used to examine the intensity and breadth of identified proteins in plasma evs purified using several density and size separation methods, either manually or automated, along with various buffer conditions. results: ev event aborts were minimized at a pfp dilution, prior to staining, of 1:10 and by using a narrow cytometer window extension. target ev signals were distinct from noise and were triton x-100 labile. the most significant changes in plasma evs were associated with platelet-derived fractions, use of heparin and >5-hour delay before blood processing. yet, platelet ev numbers did not significantly change for up to 24 hrs in citrated and edta plasma. higher overall coverage of known ev proteins and a fivefold increase in number of uniquely identified proteins were observed in ms profiling of evs prepared by a combination of ultracentrifugation (uc) and manual size-exclusion chromatography (sec) compared to preparation by fplc on capto core 700/superose 6 resins. uc/sec was better than direct sec at reducing contamination by excipient plasma proteins. column buffers with trehalose increased ev protein recovery while adding protease inhibitors had minimal effect. summary/conclusion: with our optimized hpfc protocol, we established that blood ev numbers remain stable for up to 24 hrs in acd or edta and that uc+sec with trehalose-containing buffer result in high canonical ev protein recovery. we are applying these workflows to investigate cancer-associated changes in plasma ev protein cargo. the value of exosomes as a potential biomarker for devil facial tumour disease. university of tasmania, hobart, australia introduction: the tasmanian devil (sarcophilus harrisii), the largest living carnivorous marsupial is endangered because of two transmissible cancers: devil facial tumour disease (dftd) one and two. current efforts to manage dftd are hindered by the lack of a preclinical diagnostic test for dftd. detecting dftd infection is only possible once tumours are noticed, too late to stop dftd progression. a preclinal test could tell us about unknown components of dftd pathogenesis, such as latent period and host-tumour dynamics. exosomes are extracellular vesicles released by most types of cells under both physiological and pathological conditions. exosomes have utility as diagnosis and prognosis biomarkers in a range of diseases, including cancers. the aim of this study is to investigate exosomes-based approaches towards a preclinical and progression biomarker for dftd 1 and 2 in tasmanian devils. methods: exosomes were isolated from three different dftd-1, dftd-2 and devil fibroblast cell lines by sizeexclusion chromatography. likewise, exosomes were isolated from plasma of healthy and diseased devils. to determine the size and morphology of exosomes, samples were imaged with transmission electron microscopy. exosomes isolated from cell lines and devil plasma were analysed with mass spectrometry to characterise proteins and determine their differential expression between the cell origins, and healthy and diseased animals. results: this study identified the presence of myelin proteins in exosomes from dftd cells relative to fibroblasts, which are diagnostic of dftd. additionally, we found that exosomes derived from dftd-2 abundantly express the inhibitory checkpoint molecule cd200 relative to exosomes from dftd-1 cells and devil fibroblasts, indicating a potential candidate for a differential diagnosis between tumours. moreover, exosomes from dftd cells present a greater amount of proteins related with metastasis in comparison with fibroblast exosomes, such as integrins. finally, we report the protein expression profile of exosomes from healthy and diseased devils, showing clear differences between them and the presence of immunosuppressive and metastasis proteins in animals in late stages of the disease. summary/conclusion: dftd-exosomes may provide a non-invasive diagnosis tool to detect early stages of dftd in tasmanian devils to facilitate the prevention of the disease. furthermore, dftd-exosomes may have utility as a prognosis biomarker, determining late stages of the disease using a simple a blood test, which would facilitate monitoring of wild populations. this project will provide long-term benefits for the future of the devils and encourage exosome-based solutions for other future wildlife disease outbreaks. introduction: despite the increased understanding of evs, from involvement in disease pathophysiology to therapeutic delivery, improved molecular tools to track biodistribution are largely lacking. current approaches used for ev labelling lacks sensitivity and specificity. here, we have explored bioluminescent labelling of evs to achieve a highly sensitive system for absolute in vivo quantification and tracking of exogenous evs at low cost and in a high throughput manner. methods: ev-producing cells were genetically engineered to express various tetraspanin-luciferase fusion proteins. evs purified by uf-sec from these cells were characterized by nta, multiplex bead-based array, tem and wb, followed by luciferase assay to determine the labelling efficiency. for in vitro applications cell lysate from treated cells or the conditioned medium were subjected to luciferase assay. for in vivo applications two different methodologies were applied to determine biodistribution; either by non-invasive real time in vivo imaging using ivis or by luciferase assay on harvested tissues for absolute quantification of injected evs. results: we initially performed a systematic comparison of five different luciferases for endogenous labelling of evs and identified nanoluc and thermoluc as lead candidates. we applied this technology to monitor in vitro cellular uptake and observed cell type differences in cellular uptake of engineered evs. in addition, we also observed an effect of different culturing conditions on exocytosis kinetics. for in vivo application, we applied the nanoluc labelling strategy to determine the pharmacokinetics and effect of different routes of injection on ev distribution. our results indicated a rapid uptake profile of administered evs in different tissues with liver, spleen, and lungs being the primary recipients. we also observed similar results upon tracking in vivo biodistribution in real time immediately after administration. finally, we show how different subpopulations of evs differ in their in vivo biodistribution. summary/conclusion: overall, nanoluc and thermoluc labelling of evs holds great potential for various in vivo and in vitro applications. in addition, it can enable the simultaneous detection of different subpopulations of evs in vivo, which may aid in our understanding of different sub-populations and their behaviour in vivo. apart from monitoring therapeutic evs, with one simple modification this platform offers great potential for tracking tumour derived evs both in vivo and in vitro and thus could aid in the development of anti-tumour therapies. biofunctional peptide-modified extracellular vesicles for cell targeting, macropinocytosis induction, and effective intracellular delivery ikuhiko nakase department of biological science, graduate school of science, osaka prefecture university, sakai-shi, japan introduction: [introduction] in our research group, developing therapeutic techniques based on extracellular vesicles (exosomes, evs) by effective usage of peptide chemistry to deliver therapeutic/diagnostic molecules into targeted cells has been focused. in this presentation, modification techniques using biofunctional peptides such as arginine-rich cell-penetrating peptides [1] , artificial coiled-coil peptides with receptor targeting [2] , and cell-penetrating sc18 peptides [3] derived from cationic antimicrobial protein, cap18 for cancer targeting with macropinocytosis induction, on the ev membranes will be introduced. i will also show effects of lyophilization of the peptidemodified evs on their biological activity [4] . methods: [methods] cd63 (ev marker)-gfpfusion protein expressed evs were used for cellular evs uptake assessments. all biofunctional peptides were synthesized by fmoc solid-phase method. results: [results] macropinocytosis with actin reorganization has been shown to be crucial for cellular ev uptake [5] . we developed the methods for modification of arginine-rich cpps or sc18 peptides on ev membranes using chemical linker techniques, and for example, arginine-rich cpps modification can induce proteoglycan-clustering (e.g. syndecan-4) and macropinocytosis signal transduction [1] . the artificial leucine zipper peptide-modified evs recognize the peptide-tagged epidermal growth factor receptor (egfr) on targeted cells, leading to macropinocytotic cellular ev uptake [2] . in addition, lyophilization is a useful technique for long term storage, however, we found that lyophilization negatively affected biological functions of encapsulated proteins in the evs after their cellular uptake [4] . summary/conclusion: [conclusion] these techniques and findings will contribute to development for the ev-based intracellular delivery systems. reference: [1] sci. rep. 6, 34937 (2016), [2] chem. commun. 53, 317 (2017), [3] chrmmedchem. 12, 42 (2017) [4] anticancer res. 39, 6701 (2019), [5] sci. rep. 5, 10300 (2015) os28.3 multi-compartmented microvesicles: novel extracellular secretory organelles that release exosomes and extracellular vesicles introduction: extracellular vesicles (ev) bud from the plasma membrane (pm) as microvesicles (mv) or arise from the fusion of multivesicular bodies (mvb) with the pm to release intralumenal vesicles (ilv) as exosomes. the variety of bioactive molecules carried by ev imparts diverse functionality to ev in intercellular signalling. the biogenesis and extracellular release of these specialized messenger organelles is not well understood. to investigate, we studied endothelial cells that line the inside of blood vessels, known to release ev that support angiogenesis. methods: cultured human umbilical vein endothelial cells (huvec) were examined by thin-section electron microscopy (em), serial sectioning and immunogold labelling to study the structure and composition ev release sites. to obtain optimal views of cellular ultrastructure, cells were preserved by fast-freezing and a freeze-substitution. results: a potential release site was identified in em thin sections as a discrete domain, up to several microns long, on the otherwise smooth huvec pm, where numerous bulbous membrane protrusions with thin necks were clustered. the cytoplasm in these protrusions was enriched with mvb and other vesicles and appeared to be on the verge of pinching off to release multi-compartmented mv (mcmv). consistent with this notion, in the neighbouring extracellular space, a plethora of mcmv of 300-1200 nm with ultrastructural features matching the bulbous protrusions were observed, supporting the concept that mcmv bud from the release site. serial sections confirmed that these extracellular mcmv were independent of cells and not linked by nanotubes or other processes. remarkably, fusion of mvb with the mcmv membrane was directly observed, presumably caught in the act of releasing ilv (exosomes) from the mcmv. immunogold labelling for ev markers is being used to identify proteins enriched at release sites and on released mcmv. summary/conclusion: in summary, 1) mcmv bud from localized sites on the endothelial pm, 2) mcmv contain mvb, and 3) fusion of mvb to mcmv to release exosomes occurs extracellularly. mcmv can now be evaluated as a potential source of exosome and ev release that occurs after budding from the cell of origin, adding new layers of regulation to when, where and how ev are assembled and released. funding: this work was supported by the division of intramural research of the nih. one size does not fit all: overcoming barriers to successful discovery and scaled manufacturing of therapeutic extracellular vesicles jieun lee a , wei guo b , hal sternberg c , mike west d and dana larocca d a stem cell team, seoul, republic of korea; b university of pennsylvania, philadelphia, usa; c agex therapeutics inc, alameda, usa; d agex therapeutics inc., alameda, usa introduction: extracellular vesicles have tremendous intrinsic therapeutic potential. however, the limited availability of production cell lines presents a barrier to scaled ev production and novel ev discovery. indeed, ev sources have been largely confined to a handful of cell types with the vast majority consisting of msc evs. to overcome this limitation, we developed a diverse library of hundreds of clonally pure and scalable progenitor cell lines that provides an alternative resource for ev drug discovery and production. methods: we harnessed the capacity of human pluripotent stem cells (hpsc) to differentiate into virtually any cell type by subjecting hpsc to a wide variety of media and culture conditions to maximize the diversity of partially differentiated cells. the resulting heterogeneous "candidate cultures" were plated at clonal density and further selected for self renewing and scalable clones. transcriptomic analysis indicated >200 distinct progenitor lines. cell fate potentials were mapped by screening for cell type specific marker expression in various differentiation conditions. evs were produced using cgmp methods (tff and sec) and characterized by nta, trps, surface marker analysis, rna and protein content. bioactivity assays included proliferation, migration, vascular tube network formation, senolysis, and oxidative stress. results: the progenitor library contained >200 distinct lines with diverse lineage fates including various types of bone, cartilage, muscle,and fat cells, as well as all blood vessel cell types. the lines displayed much longer replicative lifespans (70-100pd) than primary cell lines like msc. clonal purity minimized phenotypic drift resulting in maintenance of cell identity, genome integrity, differentiation capacity and bioactive ev production over extended culture. evs were highly diverse in their rna and protein cargo and bioactivity displaying various degrees of migratory, proliferative, angiogenic and senolytic activity. library screening identified evs with higher angiogenic potency than primary adult stem cell evs. summary/conclusion: we demonstrated scalable and stable production of bioactive evs from a large progenitor cell library. library screening resulted in discovery of novel angiogenic and senolytic evs having diverse rna and protein cargo. we are currently creating a corresponding library of progenitor cell evs to accelerate discovery of novel evs and their production cell lines. funding: the initial establishment of the cell library was funded in part by grants from the california institute of regenerative medicine and national institutes of health. introduction: besides extreme potential in biomedical applications, extracellular vesicles (evs) are also promising candidates to expand biophysical understanding of membrane active biomolecules. their complex bilayer composition allows the better understanding of adsorbed proteins and protein coronas as well, which sets of macromolecules will likely be key for advanced ev targeted delivery. considering cargo, membrane active peptides are interesting as these can be both drugs to be delivered, but can also facilitate cargo insertion through lipid bilayers. however, at present very little is understood regarding interactions between the peptides and the ev lipid bilayer, and between peptides and membrane associated proteins on evs. methods: we have recently demonstrated, that ev membrane adsorbed proteins and their interactions can be studied by techniques such as polarized light spectroscopy, microfluidic resistive pulse sensing measurements and freeze-fraction transmission electron microscopy [1] . furthermore, initially we studied several peptides with known antimicrobial properties and found that these strongly interact with the ev surface proteins, resulting in efficient removal of some from the lipid bilayer [2] . results: here we present investigation of further evpeptide interactions also focusing on anticancer peptides, which may be promising drug candidates for targeted delivery. these studies allowed to gain insight to novel functions of several peptides, such as melittin, magainin, buforin, lasioglossin, temporin, but also provide a more detailed understanding on how ev protein coronas, or ev bilayers are affected, to such extent that they cannot exert their potential function as delivery systems. summary/conclusion: the above interactions are expected to be interesting both for applicability, i.e. for selecting suitable compounds for ev processing, and also for curiosity-driven understanding of peptide functions, and ev-biomolecule interactions. based on these we promote that peptide -ev interactions will receive increased focus in ev-engineering. introduction: our late-breaking finding is the identification of a non-coding rna (ncrna) in extracellular vesicles (evs) from neuronal cells that is a natural antisense transcript for the dbh gene and associated with epigenetic changes and gene silencing. dna methylation in neurons is involved in memory and neurological disorders (science 2018 361 (6409)). earlier work found that during chronic brain infection with toxoplasma gondii induced a decrease in norepinephrine levels and expression of the host dbh gene; and the decrease is correlated with behaviours linked to noradrenergic signalling (infect immun. 2019 87 (2); infect immun. 2016 84 (2861)). dbh catalyzes the production of norepinephrine from dopamine in noradrenergic neurons. we found that evs from infected cultures suppress transcription of the dbh gene and hypermethylation of the gene in noradrenergic cells in vitro. in this study, we identify a ncrna in the evs from infected neuronal cells. methods: neuronal cells were induced by infection with toxoplasma gondii and evs purified on sucrose gradients. evs were characterised by electron microscopy and used to treat rat and human neuronal cells and expression levels of dbh mrna and nascent dbh gene transcription were measured. induced evs were injected into the locus coeruleus of rats and dbh gene expression was monitored. rna purified from evs was screened for natural antisense transcripts (nats) by strand-specific rt-pcr. results: we found that evs purified from infected neuronal cultures induced transcriptional gene silencing (tgs) and dna methylation of dbh in recipient neuronal cells. the induced evs down-regulated dbh gene expression >200-fold and induced dna hypermethylation of the dbh gene. this could be induced in the brains of recipient rats by intracerebral injection of evs. using a panel of strand-specific primers, antisense transcripts for the dbh gene were identified in infected cells. this permitted us to examine the rna in purified evs and identify a lncrna in evs selective for evs from infected cultures. summary/conclusion: this is the first study to find a specific neurotransmitter antisense lncrna in evs associated with transcriptional gene silencing and epigenetic changes in the gene. this represents a different type of neuron-to-neuron signalling than the classic chemical and electrical neurotransmission. the findings will enhance our understanding of neurological disorders (ie. schizophrenia, epilepsy, drug addiction) and how memory works. human cd4 + t regulatory-derived extracellular vesicles and associated micrornas: role in cell-to-cell communication and involvement in the loss of immune tolerance during multiple sclerosis introduction: an impairment of immune tolerance is a determining factor in multiple sclerosis (ms) and dysregulation of cd4 + t regulatory (treg) cell function is believed to be a major pathogenic factor. micrornas (mirnas) released by treg cells in association with extracellular vesicles (evs) have been shown to participate in the block of pathological immune responses by inhibiting the growth and cytokine production of cd4 + t conventional (tconv) cells, but the molecular mechanism is still poorly characterized. aim of the present work was to evaluate whether treg cell-derived ev-associated mirna signature is dysregulated in ms and whether this defect may play a role in the development of autoimmunity. methods: human treg cells isolated from blood of naïve to treatment relapsing-remitting ms patients and healthy controls were in vitro stimulated and released evs were isolated by size exclusion chromatography and characterized by nanoparticle tracking analysis, electron microscopy and flow cytometry. evassociated mirnas were quantified by traditional rt-qpcr and droplet digital pcr for absolute quantification. the actual ev-mediated passage of rna molecules from cell to cell was followed through rnaspecific fluorescent staining and treg-derived ev effect on tconv cell transcriptome was evaluated by rnaseq. results: in healthy conditions, the treatment of tconv cells with treg-derived evs was shown to cause the specific repression of genes involved in the proteasome-dependent proteolytic process, known to be crucial for t cell activation. in ms, treg-derived evs may have lost this capability as a direct consequence of a significantly decreased expression of mir-142-3p, able to target key factors of the proteasome system. summary/conclusion: our results unveil a novel molecular mechanism for treg-mediated maintenance of self-tolerance based on ev-associated mir-142-3p and its potential alteration in human autoimmunity. funding: fondazione italiana sclerosi multipla, fism, # 2016/r/10 and # 2018/r/4 revealing the proteome of brain derived extracellular vesicles isolated from human amyotrophic lateral sclerosis post-mortem tissues. introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterised by the deposition of misfolded proteins in the motor cortex and motor neurons. although a multitude of als-associated proteins have been identified, few have been associated with extracellular vesicle (ev) trafficking, a form of inter-cellular communication. additionally, the role of evs in als is undetermined, specifically in relation to pathogenic stress granule formation, a response to cellular stress involving aggregation of non-coding rnas and their rna binding proteins. therefore, this study aimed to determine the proteome of brain derived small extracellular vesicles (bdevs) isolated from als subjects and identify novel alsassociated deregulated proteins and their potential contributions to pathogenic pathways in als. methods: bdevs were isolated from human post-mortem als (n = 10) and control (n = 5) motor cortex brain tissues through an ultracentrifugation protocol (vella et al., 2017) . following thorough characterisation, bdevs that successfully met the minimum criteria required by the international society for extracellular vesicles were classified as evs. the bdevs subsequently underwent mass spectrometry analysis on the thermo scientific q-exactive hf with ultimate 3000 rslcnano. proteins identified to be statistically significant differentially expressed then underwent validation by western blotting. results: a panel of 16 statistically significant differentially packaged proteins were identified in the als bdevs. this included several up-regulated rna binding proteins and a down-regulated cell adhesion molecule; dhx30, stau1 and vcam1, respectively. pathway analysis revealed that the bdevs were enriched in proteins associated with stress granule dynamics, exosomal and lysosomal pathways. summary/conclusion: the identification of the rna binding proteins in the als bdevs suggests there may be a relationship between als-associated stress granules and als bdev packaging. the packaging of stress granule associated rna binding proteins into als bdevs may be an attempt by the cells to compensate for lysosomal dysfunction caused by stress granule accumulation, a feature of als. thus, these results highlight a potentially novel role for evs in the pathogenesis of als for long-term cultivation . the whole cultivation process of tissue preparation, cultivation, and cryopreservation has been established using strict serum-free conditions under a good manufacturing practice. long-term-cultivated hmnpcs retained stemness and hmnpcs have excellent differentiation efficiency into dopaminergic neurons. hmnpcs reversed impaired motor function in a rodent model of parkinson's disease (pd). based on the promising results in animal experiments, the clinical trial is under way (nct01860794). multiple-system atrophy (msa) is one of fatal neurodegenerative diseases with a combination of progressive autonomic nervous system disorders, parkinson's syndrome, and cerebellar pyramid syndrome. there are three types of msa such as msa-a, msa-c, and msa-p. in case of a msa-p type, it is difficult to diagnose due to the similarity of symptoms with parkinson's disease (pd). methods: in vitro and in vivo animal msa model were established and rotational behavioural was performed. npc cells were isolated and cultured based on moon et al. mirna sequencing (bgi) was performed and several bioinformatics analyses were done. results: based on the finding that hmnpcs exhibited therapeutic effects on pd, we hypothesize that hmnpcs will have a therapeutic effect on msa-p, where sympotoms are largely common with pd. as expected, transplanted hmnpcs survived, integrated, and differentiated in to dopamine neurons in the host brain, consequently leading to the functional recovery in the msa-p model. to further investigate the therapeutic key factors of hmnpcs in msa-p, mirna sequencing of the extracellular vesicles (evs) secreted from hmnpcs was performed. we found that mir-451a highly expressed in the npc-derived evs is one of key regulators of inflammatory response via nfkb pathway. we further experimentally demonstrated that mir-451a had anti-inflammatory effect on cells of msa-p condition such that the level of cx3cl1 expression and its receptor, cx3cr1 were both decreased in the msa-p modelled cells and in severe inflammatory environment in msa brain. summary/conclusion: our study first showed that mir-451a in hmnpcs-evs is one of key therapeutic factors for the recovery of brain damage through immuno-modulation in msa-p. introduction: oxidative insults are known to be involved in the pathophysiology of alzheimer's disease (ad). we have previously demonstrated that some blood-based redox-signature were associated to the cognitive scores in mild cognitive impairment patients and in ad (perrotte et al., 2019) . the aim of this study was (1) to evidence the presence of some oxidative markers in circulating extracellular vesicles (evs), and (2) to compare to their plasma levels. methods: plasma samples from healthy, mci and ad patients were from the memory clinic of sherbrooke (québec, canada). ad patients were stratified in three groups (moderate, mild and severe) according to the mmse and moca scores. total plasma extracellular vesicles (pevs) were isolated from plasma with the total exosome isolation reagent (invitrogen™ by life technologies inc.). pevs were then characterized by electronic microscopy, nta, dls and western blot. antioxidants apolipoprotein j, d (apo j, apod), the glyoxalase-1 and protein carbonyls were determined by western blot. results: in pevs, we found that apo d levels were higher in mci patients but not in ad patients. protein carbonyls levels were higher later, in pevs from moderate and severe ad while apo j levels were not different in pevs from the five groups of patients. in plasma, the pattern of apo j and apo d was different. the levels of apo d was not different in the five groups of patients while apo j levels were elevated in mci and in all ad groups. protein carbonyls were higher earlier from mild ad group, earlier than in pevs. the levels of the detoxifying enzyme glyoxalase-1 were higher in pevs than in plasma and were significantly decreased in early ad as compared to control subjects and mci summary/conclusion: these results demonstrate a differential regulation of redox homoeostasis in plasma and in pevs from ad patients. funding: acknowledgements: this work was supported by the chaire louise & andré on alzheimer's disease, foundation armand-frappier (cr) and cihr grant (tf). carlos j. nogueras-ortiz a , pavan bhargava b , sol kim b , francheska delgado-peraza a , peter calabresi b and dimitrios kapogiannis a a laboratory of clinical investigation, national institutes of ageing, baltimore, usa; b department of neurology, johns hopkins university school of medicine, baltimore, usa introduction: multiple sclerosis (ms) is a neurological disorder characterized by white matter demyelination and extensive synaptic pathology. recent studies have shown synaptic loss in the grey matter of ms brains in the absence of demyelinating lesions which could account for disease progression independent of demyelinating episodes. opsonization of synapses with complement components is a mechanism by which phagocytic cells normally prune synapses, but, when occurring in excess, it may underlie pathologic synapse loss. we sought to identify blood-borne biomarkers of hypothesized complement-mediated synaptic loss in ms using circulating neuronal-enriched and astrocytic-enriched extracellular vesicles (nevs and aevs). methods: nevs and aevs were immunocaptured in parallel from the plasma of 60 ms patients (45 with relapsing remitting, 15 with progressive ms) and 31 healthy controls, targeting the neuronal-specific marker l1cam and the astrocyte-specific marker glast, respectively. we measured the protein levels of preand post-synaptic proteins synaptopodin and synaptophysin in nevs using elisas and multiple complement cascade components (c1q, c3, c3b/ic3b, c4, c5, c5a, c9, factor b, factor h) in aevs using a luminex array. results: synaptopodin and synaptophysin protein levels in nevs of ms patients compared to controls were markedly reduced (2.5-fold; p < 0.0001 for both), whereas multiple complement components in ms aevs were markedly increased (c1q: 2.5-fold change; c3: 1.25-fold change; c3b/ic3b: twofold change; c5: 1.4-fold change; c5a: 1.4-fold change; factor: 1.5-fold change; p < 0.0001); differences were not observed in total circulating evs or neat plasma. strikingly, we found the nev-associated synaptopodin/synaptophysin and the aev-associated complement levels to be negatively correlated in people with ms (synaptopodin vs: c1q, r = −0.7 and p < 0.0001; c5, r = −0.6 and p < 0.0001; factor h, r = −0.46 and p < 0.0002/synaptophysin vs: c1q, r = −0.75 and p < 0.0001; c5, r = −0.65 and p < 0.0001; factor h, r = −0.52 and p < 0.0002), but not in controls. summary/conclusion: circulating evs provide markers of synaptic loss and complement activation in ms and suggest a link between astrocytic complement production and synaptic decline. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. methylglyoxal and glyoxal affect the protein cargoes in neuronal-derived extracellular vesicles introduction: advanced glycation end-products (ages) and their receptor rages are known to be involved in the pathogenesis of alzheimer's disease (ad). methylglyoxal (mg) or glyoxal (go) are the precursors of ages and particularly n-(1-carboxymethyl)-l-lysine (cml), the most abundant ages. mg induced tau hyperphosphorylation and causes hippocampal damage and memory impairment in mice. the aim of our study was to analyse the effects of mg and go on the neuroprotective, neurotrophic factors, inflammatory and neurodegenerative markers in the human cell line sk-n-sh and their release into the neuronal derived-evs. methods: briefly, sk-n-sh cells were incubated in fbs free media with mg and go (0.5 mm) for 24 hours. neuronal derived-evs (nevs) from culture media were isolated as previously described (haddad et al. 2019 ). nevs were characterized by electronic microscopy, nta and by western blot. cellular and nevs concentrations of bdnf, prgn, nse, app, mmp9, angptl-4, lcn2, ptx2, s100b, rage, dj-1 and alpha synuclein were determined by a luminex assay from r&d systems, inc. aβ1-40, aβ1-42, ptau t181 and total tau levels were measured also with luminex assay from emd millipore corp. results: we found that both ages precursors, at non toxic concentration, reduced the neuronal levels of nse with no effect on bdnf, ptrx-2, lcn-2, dj-1, on neurodegenerative markers and on cml. go decreased the levels of prgn, app, angpl-4 while the expressions of mmp-9 and angpl-4 were, respectively lower and higher in the presence of mg. mg and go greatly reduced the release of lcn-2 by neuronal cells in nevs. bdnf and prgn in nevs were reduced in the presence of go. both mg and go did not modify the release of nse, app, mmp9, agntl-4, ptx-2, dj-1, aβ, ptau and cml in nevs. summary/conclusion: our data demonstrated that mg and go differently affect the content of some protein cargoes in nevs and suggest that targeting mg and go may be a promising therapeutic strategy to prevent neurodegeneration. introduction: peripherally circulating brain-derived extracellular vesicles (evs) and their encapsulated rnas may serve as biomarkers for hiv-associated neurocognitive disorders (hand). however, rates of cigarette smoking are significantly higher in hiv+ individuals than the general population, and smoking can modulate the expression of these markers. to better understand how cigarette smoke might modulate rna expression and ev release, we examined several cnsderived cell lines, representing astrocytes (u87 mg), microglia (sv40), and oligodendrocytes (hog). methods: cigarette smoke extract (cse) was prepared by bubbling through culture medium using a standardized and published method. all cell types were exposed to either 0% or 50% cse for 24 hours. cell viability was assessed by musetm cell analyser, and evs were isolated from culture conditioned media (ccm) by size exclusion chromatography. the void (fractions 1-6), ev (7-10), and protein (11-14) enriched fractions were pooled and concentrated. evs were characterized by transmission electron microscopy (tem), microfluidic resistive pulse sensing, and western blotting. total rna was isolated from cells and circular rna (circrna) expression was assessed with a circrna microarray. results: in response to cse exposure, cell viability was only slightly reduced for all cell types. tem images validate the presence of vesicles in the ev fractions, and their absence in the void and protein fractions. spectradyne particle counts indicated cse exposure substantially increased the ccm particle count in the ev fraction when compared with control. the presence of expected ev markers (cd63, cd81, and tsg101) in the ev fractions, and their absence in the void and protein fractions was observed via western blot. intracellular circrna expression was significantly altered in all three cell lines. summary/conclusion: cns cells display physiologic responses to cse that include vesiculation pathways and significant alterations in circrna expression. we are now studying the effects of cse exposure on circrna expression in released evs. funding: this work is supported by da040385, da047807, and ai144997. a method for exosomal rna extraction from paired human brain and blood specimens emily n. moya a , lillian wilkins a , esther cheng a , lisa linares b , brian kopell b , navneet dogra c , bojan losic a and alexander charney a a icahn school of medicine at mount sinai, new york, usa; b mount sinai hospital, new york, usa; c department of genetics and genomic sciences, department of pathology, icahn school of medicine, mount sinai, new york, usa introduction: diagnosis and treatment of neuropsychiatric disorders has made little progress in the last half-century likely in large part due to the absence of a scalable technique to profile the complex biological activity of the brain in a living person. exosomes are nanovesicles 30-150 nm in size that mediate intercellular communication and contain proteins, lipids, and nucleic acids. it has been shown that brain derived exosomes can be found in peripheral blood, but determining whether peripheral exosomes truly reflect ongoing brain processes has to date not been possible due to the absence of paired living brain and blood specimens. here, we present a novel method for paired sampling of the dorsolateral prefrontal cortex (dlpfc) and peripheral blood from living human subjects for exosomal rna profiling. methods: informed consent, approved by the irb at the icahn school of medicine at mount sinai, was obtained for patients undergoing deep brain stimulation (dbs). paired brain and blood specimens were collected from 8 patients at two deep brain stimulation (dbs) electrode implantation procedures: left hemisphere followed by right hemisphere (total of 30 samples). we developed protocols to profile rna from exosomes of brain tissue extracellular matrix (ecm) and peripheral blood. exosomes were isolated via our in-house protocol using ultracentrifugation. rna was then extracted from the exosomes using the qiagen mirneasy mini kit protocol. quality control (qc) was performed to determine whether rna obtained was sufficient for next-generation sequencing. results: we demonstrate the safety of a novel procedure to sample the brain in living human subjects. bioanalyzer traces and qc data show a mean total rna of 14.83 ng (range 1.72-137.17 ng) and no samples fell below the threshold required for library preparation and sequencing (10 pg) determined by inhouse optimization on the smart-seq v4 ultra low input kit. summary/conclusion: to our knowledge, we have performed the first study to sample pairs of dlpfc and blood from living human subjects for exosomal rna for subsequent next-generation sequencing. ongoing analyses by our group promise to establish peripheral exosomal rna transcripts reflective of brain activity. this non-invasive approach to probing neurobiology in the living human brain may facilitate the development of exosome-based diagnostics for neuropsychiatric disorders. introduction: the relationship between obesity and dementia is complex. while obesity in middle age triples the risk of dementia 30 years later, many patients with alzheimer's disease (ad) are cachectic, and a decline in adiposity portends progression of dementia. this suggests adipose-derived factors are important to nervous system homoeostasis. we previously showed that adipocyte-derived small extracellular vesicles (ad-sevs) induce pathologies critical to developing obesity-related diseases and may provide a mechanistic link between adiposity and dementia. we hypothesized that altered expression of ad-sev micrornas involved in neurodegenerative pathways is associated with more severe cognitive impairment methods: we studied serum and cerebrospinal fluid (csf) from 19 participants with ad and 14 non-ad controls. ad-sevs were isolated from samples by precipitation and immunoselection. ad-sev microrna expression was profiled in both biofluids and compared. results: serum and csf microrna expression correlated strongly (r2 = 0.98). in serum, 189 micrornas were differentially expressed by a fold change ≥|1.1| in the ad and control groups (p ≤ 0.1) and 251 micrornas were differentially expressed in csf. using ingenuity pathways analysis, we identified mrnas expressed in nervous system tissue that are targeted by the differentially expressed micrornas. the mrnas map to 145 diseases and functions; neuronal cell death, neurodegeneration, and neuronal growth and developmental pathways are highly represented. of the 189 differentially expressed micrornas in serum, 6 were moderately correlated with participants' score on the mini-mental state exam, a test of cognitive function (rs = |0.488-0.609|). as validation, rencell cx cortical derived neuronal stem cells had decreased doubling time when exposed to ad-sevs from obese adipose tissue in vitro. summary/conclusion: these findings support our hypothesis that altered expression of circulating ad-sev micrornas are involved in neurodegenerative pathways associated with cognitive impairment. these findings support using serum ad-sevs as a surrogate for csf ad-sevs. functional validation is underway to define the connection between ad-sevs and ad. understanding the link between obesity and ad is crucial as the population ages and the global obesity epidemic grows. funding: supported by uw adrc (nih:p50ag005136) expression of extracellular vesicles after acute traumatic brain injury: an exploratory flow-cytometry study introduction: coagulation derangements related to disseminated intravascular coagulation (dic) are common after tbi and contribute to secondary neural injury. extracellular vesicles (evs) are released from all cell types, including platelets, endothelium, and lymphocytes, which are responsible for dic. we hypothesized that specialized flow cytometry techniques could identify a unique ev signature of dic in acute tbi. methods: using a modified flow cytometry instrument for detection of small particles, fluorescence panels were created to assess for evs from endothelial cells (cd144, cd105), platelets (cd31, cd62p, cd41a, cd42b), and erythrocytes (cd235) as well as brain biomarkers (s100b, uchl-1, gfap, tau and nse) and t-lymphocytes (cd3, cd4, cd8, cd31). samples were prepared in trucount tubes to determine volume and treated with triton to confirm presence of evs. results: 13/17 study patients and 16/20 controls were male. 76% of study patients presented with a glasgow coma scale of 15. in the hypercoagulability panel, of the 10 subsets with statistically significant differential expression, 4 involved s100b+ and were elevated in patients. platelet-derived cd41a evs and uch-l1 evs were significantly elevated in controls in 7 ev subsets identified in the brain-specific panel. finally, cd3+/31+ evs, derived from t-cells and identified in the endothelial/t cell panel, are significantly lower in patients suggesting cns recruitment. summary/conclusion: endothelial and platelet/erythrocyte evs may be elevated early after tbi. s100bcarrying evs are significantly elevated in circulation of tbi patients; if reproducible, this signature profile may be informative for diagnosis and risk stratification. further study is warranted to evaluate whether this expression correlates with secondary microvascular brain injury. funding: intramural award from the university of pennsylvania enrichment of mir-451a in cns extracellular vesicles following impairment of the blood brain barrier nasser nassiri koopaei a , ekram-ahmed chowdhury b , lais da silva a , jinmai jiang a , behnam noorani b , ulrich bickel b and thomas d. schmittgen a a university of florida, gainesville, usa; b texas tech university, amarilo, usa introduction: extracellular rnas (exrnas) are present in essentially all biofluids and include all types of rna including mirna. to enhance their stability outside of the cell, exrnas are bound within ribonucleoprotein complexes or packaged into extracellular vesicles (evs). the blood brain barrier (bbb) is a dynamic interface between the systemic circulation and the cns and is responsible for maintaining a stable extracellular environment for cns cells. the intent of this study was to determine if evs and their contents are transferred from the peripheral circulation to the cns under conditions of an impaired bbb. methods: the bbb of mice was disrupted by hyperosmolar mannitol injections. to validate that the bbb has been disrupted with mannitol, intravenously-dosed [13 c]-sucrose was increased in the forebrain by 14fold with mannitol compared to sham treated mice. evs were isolated from the forebrain, hindbrain and spinal cord following gentle tissue lysis and differential ultracentrifugation. evs were validated by nta, tem and western blotting. mir-451a, a mirna that is highly abundant in erythrocytes, was measured in the evs by qpcr. results: qpcr showed that mir-451a in cns tissue evs increased with mannitol treatment in the forebrain, hindbrain and spinal cord by 15-, 1.6-and twofold respectively. qpcr analysis of mrna from reported mir-451a target genes showed reduced target gene expression with mannitol. summary/conclusion: we demonstrate that evs containing mir-451a, a highly abundant mirna present within erythrocytes and erythrocyte evs, is enhanced in the cns upon bbb disruption. astrocyte-derived extracellular vesicles in morphine tolerance guoku hu, rong ma, naseer kutchy, yuetong zhao, susmita sil and shilpa buch university of nebraska medical center, omaha, usa introduction: opiates, such as morphine are used extensively in the clinical setting owing to their beneficial effects. paradoxically, however, the prolonged use of morphine often results in the development of tolerance, drug addiction, and ultimately leading to various comorbidities associated with drug abuse. although great efforts have been made, at present there is no treatment. the sonic hedgehog (shh) plays a key role in brain development, and brain cells fine-tuning processes such as their proliferation, patterning, and fate specification recent findings have demonstrated that inhibition of the shh signalling prevents morphine tolerance in rodent models. we thus hypothesize that extracellular vesicles (evs) derived from morphine exposed astrocytes and their cargo such as shh are critical for the development of morphine tolerance. methods: mice were received either saline or chronic morphine injection with escalating doses of morphine for 5 days (subcutaneously; 10 mg/kg, day 1, 20 mg/kg days 2-3, and 40 mg/kg days [4] [5] . the development of tolerance was assessed by measuring the tail-flick latency using tail flick analgesia metre (le7106, harvard apparatus). evs were isolated using either differential ultracentrifugation from astrocyte conditioned media or gradient ultracentrifugation from brain tissues. western blotting and qpcr were performed to determine the expression/activation of shh signalling pathway components. results: our data showed that the levels of shh protein were upregulated in morphine exposed astrocytederived extracellular vesicles (morphine-adevs). furthermore, shh containing morphine-adevs activated shh signalling in astrocytes. our in vivo study further demonstrated the upregulation of shh, as well as the activation of shh signalling, in astrocytes of morphine-administered mice. summary/conclusion: these findings thus demonstrated an autocrine mechanism for shh pathway activation in astrocytes associated with morphine tolerance. these findings could pave the way for the development of shh signalling pathway targeted strategies in the prevention and treatment for substance use disorders. biophotonics-based platforms for the evaluation of circulating extracellular vesicles as biomarkers of neurodegeneration in alzheimer's disease silvia picciolini a , cristiano carlomagno a , alice gualerzi a , monia cabinio a , francesca baglio a and marzi bedoni b a irccs fondazione don carlo gnocchi, milan, italy; b irccs fondazione don carlo gnocchi, milano, italy introduction: in the search for novel and non-invasive biomarkers of alzheimer's disease (ad), both circulating brain-derived extracellular vesicles (evs) and whole serum represent a valuable integration of the currently used classification system. to face the technological challenge of evs and serum analysis, we propose the use of biophotonics techniques as reliable, sensitive, fast and label free methods, potentially useful in tailoring pharmacological and rehabilitation treatments. methods: circulating evs, isolated by sec, and serum samples were collected from 10 healthy subjects (hc) and 10 ad patients. all subjects were asked to complete montreal cognitive assessment scale and mri examination. surface plasmon resonance (spr) was performed in order to detect evs coming from neurons, astrocytes, oligodendrocytes and microglia and to characterize each of them for the amount of ganglioside m1 (gm1), aβ and tspo expressed on their surface. serum analysis was performed using a raman microscope through the surface enhanced raman spectroscopy (sers) effect by mixing serum with ag nanoparticles. the pearson's correlation index was used to assess the linear correlation between spri data and clinical, mri data and data obtained from multivariate analysis (mva) of sers spectra. results: the spr analysis of evs showed that the selected bioactive molecules are differently loaded on neural ev populations and that their amount is increased on total evs in ad patients compared to hc. we observed a significant correlation between mva data from sers and the presence of aβ on neuronal and microglial evs and of tspo on neural evs, measured with the spr array. summary/conclusion: thanks to our methodological innovation we have verified the potentiality of evs as ad biomarkers, correlating biophotonics blood-based analysis with clinical data. this platform could provide a powerful tool for the evaluation of ad neurodegeneration. funding: the study was supported by the italian ministry of health (ricerca corrente 2017-2018 to irccs fondazione don carlo gnocchi). raman profiling of extracellular vesicles as new blood-based biomarker for brain disorders: focus on parkinson's disease introduction: extracellular vesicles (evs) play a pivotal role in brain homoeostasis and intercellular communication in both physiological and pathological conditions. in parkinson's disease (pd), evs are key players in the transfer of α-synuclein, with blood evs reported to undergo proteomic modifications. nonetheless, the detection and characterization of the ev cargo is technologically challenging, limiting the use of evs as biomarkers so far. herein, we propose raman spectroscopy for the label-free, bulk characterization of blood evs in pd patients. methods: evs were isolated by sec and ultracentrifugation from the serum of 18 healthy subjects (hc) and 22 pd patients. in all patients, the severity of pd was evaluated with the unified parkinson's disease rating scale (updrs) part iii and with hoehn and yahr scores (hy). after proper ev characterization following misev2018 guidelines, raman analysis was performed. the raman microspectroscope was used with a 532 nm laser in the spectral ranges 600-1800 cm-1 and 2600-3200 cm-1. data from hc and pd patients were compared by multivariate statistical analysis (pca-lda). results: the raman analysis of evs highlighted differences in the biochemical profile of the two groups, with the main variations in the spectral regions related to proteins, lipids and saccharides. a preliminary estimate of the accuracy of raman profiling of blood evs for pd diagnosis was obtained, demonstrating an accuracy of 71%. even more interestingly, we demonstrated the correlation between the raman spectra and the clinical scales (updrs and hy) used to stratify pd patients. summary/conclusion: in conclusion, the biochemical signature of blood evs can be detected by raman spectroscopy in pd patients and the ev spectral modifications can be related to their clinical status. these data suggest the possibility to use the raman profile of circulating evs as a biomarker for brain disorders, complementary to other specific molecular markers. funding: the study was supported by the italian ministry of health (ricerca corrente 2017 to irccs fondazione don carlo gnocchi) impact of circulating extracellular vesicles on brain functions and behaviours introduction: peripheral immune alterations have been described in psychiatric disorders such as schizophrenia, depression, and autistic spectrum disorders. in addition, behavioural changes have been observed in various immunodeficient animal models. however, the mechanisms by which peripheral immune system influences brain development and function are not well understood. in this study, we explored the mechanisms by which circulating extracellular vesicles (evs) mediate immune-brain communication and influence mouse behaviours. methods: mice deficient for rag1 or rag2 gene (rag ko mice) were used as a model to study the effects of loss of adaptive immune cells (t and b cells) on brain cellular phenotypes and behaviours. circulating evs were collected from their sera and analysed by using electron microscopy, nanoparticle tracking assay, and western blotting. brain cellular phenotypes were assessed by immunofluorescent staining and gene expression analysis. behavioural phenotypes of rag ko and wt mice were examined in social interaction test. in vivo transfer of evs was performed to see its effects on behavioural alterations of rag ko mice. results: rag ko mice displayed social behavioural deficits, accompanying by enhance c-fos immunoreactivity and altered microglia morphology in the medial prefrontal cortex (mpfc). circulating evs were also affected in these mice and lacked the expression of markers for t cells. a set of micrornas (mirnas) in circulating evs were diminished in rag ko mice. in vivo transfer of circulating evs rescues the social behavioural deficits of rag ko mice and ameliorate the c-fos immunoreactivities in mpfc of rag ko mice. summary/conclusion: our data showed that circulating ev profiles were altered in mice lacking adaptive immune cells and, accordingly, showing social behavioural deficits. notably, our in vivo experiments suggest that circulating evs may contribute to social behaviours. further study will provide a novel biological insight into the mechanisms underlying peripheral-to-brain immune communication via evs. introduction: the involvement of neuroinflammation on ageing process is widely recognized. extracellular vesicles (evs), such as exosomes, are able to cross the blood-brain barrier and were related to neuroinflammation. in this context, evs have been considered a potential mechanism of spreading molecules, including micrornas (mirnas) that can promote mrna degradation or inhibit translation of their targets. our aim was to investigate the mirna profile of circulating total evs during ageing process and their impact on canonical pathways. methods: the local ethics committee (comissão de ética no uso de animais -ufrgs; n 29818) approved all animal procedures and experimental conditions. plasma was obtained from wistar rats (3 and 21 months-old) and total evs were isolated. ev microrna isolation and microarray expression analysis was performed to determine the predicted regulation of targeted mrnas. results: the analysis of global microrna expression revealed 48 differentially expressed micrornas (p < 0.05; fold change of ≥ |1.1|); 18 mirnas were up-regulated and 30 were down-regulated in circulating total evs from aged animals compared to youngadult ones. a conservative filter was applied on ingenuity pathway analysis (ipa) and only experimentally validated and highly conserved predicted mrna targets were used. ipa showed that neuroinflammation signalling is ranked among the top canonical pathway impacted by differentially expressed micrornas and is upregulated in aged animals (p < 0.0001; z-score: 3.413). the differentially expressed mirnas impacted 32 molecules in the neuroinflammation pathway. interestingly, the ion channel grin2b is predicted to be up regulated and is a target of many evs mirnas; in accordance with our results grin2b was previously related to neurodegenerative diseases. moreover, let-7a-5p is predicted to be downregulated and target all the 32 molecules of the neuroinflammation signalling pathway. previous studies have correlated let-7a-5p and neurodegenerative diseases. summary/conclusion: our data suggest that circulating total evs cargo, specifically mirnas, are altered by ageing and impact neuroinflammation pathway, suggesting the involvement evs mirna on ageinginduced susceptibility of neurodegenerative diseases. introduction: bidirectional cell-cell communication via paracrine mechanisms is critical for wound healing. a new paradigm involving exosome-borne distinctive repertoire of cargo such as mirnas has emerged as a predominant mechanism of cellular communication at the site of injury. unlike other shedding vesicles of similar size, exosomes selectively package mirna by sumoylation of heterogeneous nuclear ribonucleoprotein (hnrnp). methods: keratinocyte-derived exosomes (exoκ) were genetically labelled with fluorescent reporter (gfp) using tissue nanotransfection. purified, gfp-labelled exoκ were isolated from dorsal murine skin and wound-edge tissue by differential ultracentrifugation followed by affinity selection using magnetic beads. distributions of intact exosome were analysed using a prototype jarrold-geometry charge-detection mass spectrometer to directly measure differences in particle mass and charge distributions. complementary ms and ion mobility spectrometry (ims)-ms experiments have been used to characterize surface glycans and glycopeptides. to selectively inhibit mirna packaging within the exoκ in vivo, ph-responsive targeted sirna functionalized lipid nanocarriers (tlnκ) were designed using materials that have prior history of fda approval for human use. results: an increase in mass/charge ratio with glycan binding sites on the surface of wound-edge exoκ were observed compared to dorsal skin exoκ. wound-edge exoκ were selectively taken up by the macrophages in the granulation tissue (n = 6). keratinocyte targeting sirnahnrnp functionalized lipid nanocarriers (tlnκ) were designed with encapsulation efficiency of 94.3%. application of tlnκ encapsulating sirna of hnrnp (tlnκ/si-hnrnp) to murine dorsal woundedge significantly inhibited the expression of hnrnp by 80% in epidermis compared to control (tlnκ/sicontrol)(n = 10). moreover, mice treated with tlnκ/si-hnrnp showed impaired barrier function, with significant presence of macrophage in granulation tissue at day 10, suggesting impaired conversion of macrophage in the granulation tissue. summary/conclusion: this work provides a novel insight wherein exosomes of keratinocyte lineage are recognized as a major contributor that directs macrophage conversion in granulation tissue for wound healing. multifaced effects of milk-exosome (mi-exo) as modulator of scar-free wound healing gna ahn, hyo-won yoon, yang-hoon kim and ji-young ahn chungbuk national university, cheong-ju, republic of korea introduction: recently, milk exosome (mi-exo) has been focused particularly on the possibility of oral distribution for therapeutic agents. however, studies related to the cosmeceutical effects associated with mi-exo are fairly limited. the purpose of this study is to suggest the anti-oxidant and antiinflammatory effect of mi-exo and possibility that can be induced by scar free healing by micro rna in mi-exo. methods: the characteristics of the extracted mi-exo were verified by size measurement, morphological characteristics through cryo-em and western blot. for antioxidant experiments, an abts assay was performed. next, mrna expression through four major cytokines (tnfα, il-6, cox-2, inos) was used to evaluate anti-inflammatory effects. finally, cell migration assay was performed to confirm the effect of scar-free healing and the detection of mir-30b in mi-exo and vegf mrna expression confirmed. results: mi-exo using 1% acetic acid extraction showed the highest yield. the average size of the exosomes is approximately 110 nm, confirmed the typical double membrane vesicle. as a result of antioxidant experiments, it was confirmed that the treatment of exosomes of 10^10 particles showed about 65% antioxidant activity. when 10^10 particles were treated, rna expression of cytokines showed about 2 times more inhibitory effect than control. elisa test results also confirmed that the concentration-dependent decrease. the activation of the raw cell less proceeded as the treated mi-exo increased. the cell scratch assay cells did not close the cells as the number of milk exosomes increased (wound closing % of 10^10 particle = 4.7%). and mir-30b in milk exosomes was detected at ct value = 22. summary/conclusion: the antioxidant and antiinflammatory effects of mi-exo showed the greatest efficacy when 10^10 particles were treated. in addition, it induced to scar free healing rather than wound healing. mi-exo has great potential as a superior natural material in the future cosmeceutical field. extracellular vesicles in human milk expose tissue factor and promote coagulation introduction: tissue factor (tf), a transmembrane protein, initiates coagulation by binding and activating coagulation factor vii (fvii). tf is associated with extracellular vesicles (evs) in saliva and urine, but it is unknown whether also human milk (hm) contains evs exposing coagulant tf. methods: hm was collected from six healthy nursing women with informed consent. evs were isolated by ultracentrifugation and size exclusion chromatography (sec). the presence of tf antigen exposing evs was studied by western blot, flow cytometry, cryo-electron microscopy (cryo-em), and surface plasmon resonance imaging (spri). the ability of tf exposing evs to trigger coagulation was investigated with a plasma fibrin generation test (fgt), performed in the absence or presence of antibodies against tf or fvii(a). results: addition of hm to plasma shortened the plasma clotting time, even when hm was highly diluted. after ultracentrifugation of hm, both tf antigen and tf activity were detected in the ev-containing pellet. after sec, tf antigen and tf activity were present in the ev-containing fractions 8 and 9. the presence of tf-exposing evs in these sec fractions was confirmed by western blot (cd9, cd63 and tf), flow cytometry, spri, and fgt. in addition, the presence of evs in hm was confirmed by cryo-em. scalable isolation of evs from different probiotic strains with potential as cosmetic ingredients laura soriano-romaní, joaquin espí and begoña ruiz ainia, paterna, spain introduction: extracellular vesicles (evs) are increasing their application in a number of fields. recently, it has been shown that skin health may be affected not only by commensal skin bacteria, but also by the evs that they secrete. however, because most of the efforts have been directed to the characterization and evaluation of evs, the scaling up of the production process remains a bottleneck at the industrial level. in this work, the goal was to evaluate the potential applications of evs produced by different probiotic strains commonly used in the cosmetic field, considering the economic and technical viability of the process. methods: to meet our goal, a standardized workflow was defined to isolate evs from probiotic strains such as lactobacillus and bifidobacterium species, that have demonstrated cutaneous immuno-regulatory effects. the different bacterial strains were produced under standard culture conditions. to isolate the secreted bacterial evs, different chromatographic techniques were performed starting from clarified growth medium. then, evs were evaluated in vitro for a number of biological effects related with skin health. results: the ev yields obtained after downstream processing were calculated for each strain and isolation technique by means of nanoparticle tracking analysis (nta) and total protein content. moreover, evs were visualized by electron microscopy. the in vitro evaluation of isolated evs was based on changes in the expression of five biomarkers related with anti-ageing, anti-inflammatory and whitening effects using distinct skin cell types to identify possible cosmetic claims that could be associated to each probiotic source. summary/conclusion: the potential of evs obtained from probiotic strains as cosmetic active cell-free ingredients was preliminarily assessed with this work, where the process yield and cosmetic function were evaluated. however, additional experiments will be needed in order to increase and optimize the productivity of each step of the ev manufacturing process. acerola derived exosome-like nanovesicles enhances the repair of ultraviolet b-induced dna damage in cultured skin fibroblasts tomohiro umezu, masakatsu takanashi, yoshiki murakami and masahiko kuroda tokyo medical university, shinjyuku, japan introduction: acerola (melpighia emarginata dc.) is a fruit is known to contain not only high amounts of ascorbic acid but also various nutritional components such as carotenoids and polyphenols. previous reports showed the acerola juices are able to confer protection against ultraviolet radiation b (uvb), to improve barrier function of skin. uvb is the main cause of dna damage in epidermal cells, generating several types of pro-mutagenic lesions, like cyclobutene prymidine dimers (cpds) and prymidine (6-4) prymidinone photoproducts (6-4pps): if not repaired, this dna damage leads to skin cancer. in this study, we investigated the biological property of the acerola derived exosome-like nanovesicles (adens), aiming to clarify the involvement of adens in repair of uv-induced dna damage. methods: normal human dermal fibroblasts (nhdfs) were purchased from lonza inc. the exosome-like nanovesicles were isolated from acerola juices using exoeasy maxi kit (qiagen). the morphology and size distribution of adens were checked by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta, nanosight lm10, malvern). nhdfs were exposed to uvb (1 mj/cm2) with pre-or post-adens. effect of uvb was assessed by examining cell viability, cell morphology, and dna damage levels through biochemical assays, microscopy and protein expression studies. results: purified adens were compatible with nta or tem for assessing the nanovesicle size range and concentration (200-400 nm). when nhdfs were added with adens and incubated at 37°c for 48 h, there was no effect of adens on cell proliferation of nhdfs. we found that adens treatment to uvb exposed nhdfs significantly reduced cpds and 6-4pps dna adduct formation. present results showed that aden treatment prevented uvb induced dna damage in nhdfs. summary/conclusion: we confirm that adens have the effect of repairing dna damage caused by uvb. these results provide that adens can be a new source to protect human skin from uv-induced skin cancer. introduction: introduction: despite the development of a variety of therapies, complex wounds resulting from disease, surgical intervention, or trauma remain a major source of morbidity. extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) have been shown to improve wound healing, especially via enhanced wound angiogenesis. however, despite their clearly established potential, evs have limitations that may limit clinical relevancy, such as low potency. hypothesis: increased expression of pro-angiogenic lncrna hotair within msc evs enhances their proangiogenic effects and thus their wound healing properties. methods: methods: hotair was overexpressed in human dermal microvascular endothelial cells (hdmecs) to determine any molecular or functional pro-angiogenic effects. anti-angiogenic mirnas and angiogenic mrna levels were quantified by rt-qpcr. effects of hotair on proliferation of hdmecs was also determined. hotair was then loaded into msc evs by delivering a cmv-based hotair plasmid to mscs for endogenous loading via a concentration gradient. evs were collected by differential centrifugation. hotair content within evs was confirmed by gel electrophoresis and rt-qpcr. effects on migration of hdmecs by hotair-loaded msc evs were determined using a scratch assay. results: results: overexpression of hotair decreased mir-29 c and mir-107, while increasing vegf and hif-1a. hdmec proliferation was also increased in hdmecs overexpressing hotair (p < 0.01). hotair was visually confirmed in hotair-loaded msc evs by gel electrophoresis, but was undetectable in unmodified msc evs. rt-qpcr confirmed a 900-fold increase of hotair compared to control msc evs. hdmecs showed a more statistically significant rate of gap closure when treated with hotair-loaded evs (p < 0.01) than compared to control msc evs (p < 0.05). summary/conclusion: summary: loading lncrna hotair into msc evs is achievable by a concentration gradient-dependent method and offers potential to enhance the angiogenic properties of msc evs. nanomaterial labelling of exosomes for cell biology introduction: exosomes are vesicles secreted by many, if not all, cell types and have been known about for decades. among larger micro vesicles that are produced directly from the cell membrane, the small (30-150 nm), exosomes are similar in size to a virus surrounded by a lipid bilayer. we and others have demonstrated that exosomes contain proteins, lipids, rna, and dna, making them promising materials for diagnosing and treating diseases, including many cancers such as brain cancer. in addition, exosomes from neurons and glial cells represent a novel type of intercellular communication. however, their size makes them hard to track with traditional fluorescence microscopy. to address this, we developed photothermal microscopy (ptm), which uses gold nanomaterial labelling to track exosomes' interaction with and effect on cells/tissue. methods: exosomes secreted by tumour cells and general exosomes found in the blood were isolated using differential ultracentrifugation or a commercially available kit (invitrogen). next, the exosomes were characterized by (tem), (nta), and western blotting to determine shape, size, morphology and the protein profile in the exosomal membrane. after characterization, the exosomes were labelled with gold nanoparticles via sonication. next, the samples were washed, and the exosomes were labelled with fluorescence dye to stain the membrane. after staining and labelling, the exosomes were added to u87 cells in culture and incubated for 3 h. they were then fixed by 4% paraformldehyde and imaged by ptm. results: ptm found that exosome-cell interactions are exosome-type dependent, as u87 cells took up exosomes from other u87 cells but not human serum exosomes. this suggests that exosome uptake is a selective process and depends on the source of the donor cells. summary/conclusion: exosomes can be labelled with gold nanoparticles via sonication then successfully tracked by ptm to study the effect of exosome source on exosome-cell interactions and communication. cells incubated with u87 exosomes took the vesicles up rapidly, while cells incubated with serum exosomes had little uptake. ptm will help us design selective exosome-based strategies to treat different conditions, including brain cancer and cns damage. funding: nsf epscor riii award 1457888. loading of goat´s whey extracellular vesicles with spiked microrna and curcumin as an strategy for developing new nanocarriers for acellular therapies introduction: extracellular vesicles (ev) are involved in cell signalling and are present in a variety of cell secretions such as milk, from which enormous amount of ev can be purified, thus milk is an attractive raw material for scaling up ev production for therapeutic, cosmetic or other uses. here we isolated evs from the whey fraction of goat´s milk and demonstrated that such evs can be loaded with molecules like polyphenols and mirna. methods: to achieve this, milk was collected from lactating goats and fractionated by acidification and centrifugation into whey and caseins. evs were purified from the former fraction by serial centrifugation and precipitation with commercial kit (total isolation/ thermo fisher) and characterized by electron transmission microscopy (tem), western blot to identify surface markers and measurement of size through nanotracking analysis. once isolated, evs were loaded with different concentration of a spiked synthetic mir39 or with the polyphenol curcumin. mirna or curcumin were co-incubated over night with evs at 4oc, precipitated and purified as described above, with an additional washing and precipitation for curcumin. concentration of mirna uploaded by evs was quantified using mir39 specific qpcr. curcumin was measured using a spectrophotometer at 420 nm. results: evs isolated from whey had an average size of 120 nm, were positive for hsp70, cd9 and alix. in tem, evs were identified with their natural conformation and corresponding size to exosomes. qpcr showed a significant difference of expression of mir39 in relation to control (loaded with shame) and the negative control (p < 0.05). curcumin presence was also confirmed after washing and precipitacion. summary/conclusion: in conclusion, milk evs and exosomes can be loaded with mirna and a polyphenol and can be used as alternative nanocarrier for acellular therapies. introduction: extracellular vesicles (evs) are cellderived lipid membrane nanoparticles that serve as messengers of intercellular communication, transferring bioactive molecules to recipient cells. evs have a natural therapeutic potential with high flexibility and biosafety for employing natural and synthetic biomolecules as therapeutic delivery vehicles. considering the importance of evs, their isolation methods are still a bottleneck. to get insights into the tissue-specific cargo in vivo for complete exploitation of evs as therapeutic, biomarker and diagnostic tools, ev purification methods are critical. the aim of the study was brought about to develop an efficient ev purification method both in vitro and in vivo and to further investigate function of evs in cellular senescence. methods: to isolate tissue-specific evs in vivo we developed recombinant evs by genetically fusing snorkel-tag to the cd81. the snorkel-tag enables on-column protease treatment for purifying evs which does not rely on traditional immunoaffinity purification protocols using low ph or high salts solutions. results: we systematically evaluated the purification of evs harbouring snorkel-tag by employing different methodologies. our findings suggest that evs harbouring snorkel-tag indeed can be purified at high purity without altering ev biophysical properties. furthermore, we expressed cd81-snorkel-tag under p16ink4a promoter and were able to purify evs derived from senescent cells. summary/conclusion: finally, we are developing an in vivo model with cd81-snorkel-tag under p16ink4a promoter. this will provide us detail insights into the ev cargo secreted from senescent derived cells, by purifying evs harbouring snorkel-tag under pathophysiological conditions, allowing us to develop biomarkers and therapeutic tools. summarized, we have here developed novel tool for studying content and function of evs in the context of ageing and disease. this tool will now pave the way for studying the molecular mechanisms underlying these ev functions in vivo. funding: this work was funded by the austrian science fund phd program biotopebiomolecular technolgy of proteins (w1224). engineering exosomes with gata-4 jie xu, christian paul, yi-gang wang and meifeng xu university of cincinnati, cincinnati, usa introduction: exosomes, are small vesicles (30-150 nm) secreted from cells that can transport and deliver of their components such as lipids, proteins, dna, mrna, and mirna to target cells. gata-4, a cardiac transcription factor, has been shown to regulate differentiation, proliferation, and survival of a wide range of cell types. delivering gata-4 protein into ischaemic tissues may be one of the most straightforward approaches to improve cardiac function following myocardial infarction. here, exosomes were engineered with gata-4 by infusing gata-4 with exosome targeting peptide. methods: the open reading frame of mouse gata-4 cdna was ligated to xpack lentivirus vector (xpack-gata-4) and plvx-ef1-ires-pouro lentivirus vector (plvx-gata-4), respectively. hek 293 cells were transduced by lentivirus, then exosomes were isolated from conditioned medium of hek293 cells using ultracentrifugation. exosomes were identified using transmission electronic microscope (tem), and the expression of gata-4 was semi-quantified using western blot. the internalization of exosomes was tracked via treating bend3 cells with exosomes pre-labelled with pkh26. introduction: chinese hamster ovary (cho) cells have dominated as the mammalian cell host for the manufacture of humanized biologics, in part owing to their genomic plasticity and robust growth in suspension culture. there is great interest surrounding the use of extracellular vesicles (evs) as novel therapeutics owing to their capacity to deliver bioactive molecules. however, much remains unknown about the mechanisms involved in ev cargo loading, limiting their development as novel biologics. to this end, we have engineered cho cells to stably express constructs enabling loading of gfp into evs. methods: tetraspanins are established markers of ev identity. accordingly, cd81 was selected as a tethering point to generate evs with gfp cargo and constructs were generated via golden gate assembly. cho cells were stably transfected by electroporation and expression was verified with fluorescence microscopy and western blotting. growth in batch culture was monitored to establish maximum viable cell densities for ev harvest and recovered evs were characterized by nanoparticle tracking analysis (nta). finally, uptake of gfp-evs was studied using time-lapse fluorescence imaging in co-culture experiments. results: strong localization of cd81-gfp was observed at the cell membrane and blotting confirmed intact tetraspanin fusion present at the expected molecular weight. additionally, cells were confirmed to retain high gfp expression post-cryopreservation. stable cell pools were able to reach viable densities greater than 7 million cells/ml in batch culture and nta allowed for detection of gfp cargo even prior to ev isolation. evmediated transfer of functional gfp to recipient cells was found to occur over a period of hours. introduction: extracellular vesicles (evs) are considered promising for therapeutic applications. evs resemble the cell membrane, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. despite the promising potential of evs for therapeutic applications, robust manufacturing processes that would increase the scalability and consistency of ev production are still lacking. methods: in this work, evs were produced by mesenchymal stromal cells (msc), isolated from different human tissue sources (bone marrow, umbilical cord matrix and adipose tissue). msc were selected as these cells allow for a scalable production of evs, while displaying low immunogenicity. a vertical-wheel™ bioreactor system was implemented for the production of msc-derived evs and compared with traditional static systems. the obtained ev products were characterized by nanoparticle tracking analysis, atomic force microscopy, zeta potential and western blot. results: the bioreactor system allowed to obtain evs at higher concentration and productivity, as well as more homogeneous size distribution profiles, when compared to traditional static culture systems. functional studies were performed using breast cancer and lung cancer cell lines. proliferation assays allowed to determine the dose-response profiles of these cell lines when exposed to msc-derived evs. a bell-shaped profile was observed for most cases, since raising the ev concentration lead to increased cell proliferation until a certain point (25-50 µg/ml), after which cell proliferation was attenuated with increasing ev concentrations. summary/conclusion: the bioreactor culture system allowed a substantial improvement in the production of msc-derived evs, while the obtained dose-response profiles will be valuable to determine the most appropriate ev concentrations for anticancer drug delivery. overall, we demonstrate that this culture system is able to robustly manufacture human msc-derived evs in a scalable manner towards the development of novel therapeutic products such as anticancer drug delivery systems. biodistribution and cellular location of inhaled exosomes and liposomes in the lung introduction: increasing evidence reveals the potential role of extracellular vesicles, such as exosomes and liposomes, in lung regenerative medicine for the treatment of lung diseases. encapsulation and delivery of potential rna and microrna targets into liposomes and exosomes are attractive drug delivery methods, but remain difficult to deliver to the pulmonary parenchyma to reach target lung cell types. here, we demonstrate effective delivery and cellular uptake of exosomes and liposomes to the pulmonary parenchyma via inhalation treatment in a murine model of idiopathic pulmonary fibrosis. methods: human lung stem cells (lscs) were generated and expanded from healthy whole lung donors. lsc-exosomes were purified via ultrafiltration and diilabelled using vybrant☐ labelling solution according to the manufacturer's instructions. dsred-labelled liposomes were generated using lipofectamine™ rnaimax transfection reagent and block-it™ alexa fluor™ red fluorescent control according to the manufacturer's instructions. lsc-exosomes and liposomes were delivered via nebulization to cd1 mice with bleomycin-induced pulmonary fibrosis. exosome and liposome delivery and biodistribution were visualized 4-and 24-hours post-treatment through histological analysis. the study was approved by the institutional animal care and use committee of north carolina state university and complied with all national and state ethical standards. results: exosome and liposome delivery to the pulmonary parenchyma was confirmed by the presence of dii and dsred fluorescence in lung histological sections that penetrated the mucus-lined respiratory epithelium. more exosomes and liposomes surpass mucus-lined surfaces 24-hours post-treatment compared to 4-hours post-treatment. fluorescent colocalization of exosomes and liposomes with alveolar type i cells, alveolar type ii cells, basal lung cells, and cd68 + macrophages was observed through immunohistochemistry analysis. more exosomes and liposomes colocalize with these cell types 24-hours post-treatment compared to 4-hours post-treatment. summary/conclusion: lsc-exosomes and liposomes penetrate the mucus-lined respiratory epithelium and reach the pulmonary parenchyma through inhalation treatment. lsc-exosomes and liposomes are uptaken by alveolar epithelial cells, basal cells, and interstitial macrophages with improved biodistribution 24-hours post-treatment. funding: this study was supported by the nc state chancellor's innovation fund. transfection reagent artefact accounts for some reports of extracellular vesicle function codiak biosciences, cambridge, usa introduction: extracellular vesicle (ev) functions are frequently investigated by transiently transfecting cells with plasmid dna to produce evs modified with protein(s) or nucleic acid(s) of interest. however, evs and the dna-complexes used to transduce cells are physically similar, raising the possibility that they may co-purify during differential ultracentrifugation, the most common ev isolation procedure. activities attributed to evs may therefore be due to contaminating dna -transfection reagent complex. methods: ev producing cells were transiently transfected with plasmid dna encoding gene-editing or split enzymes fused to ev-targeting protein sequences. differential and density gradient ultracentrifugation were used to purify evs from cell culture supernatant or dna lipoplexes from cell-free culture media. protein expression and localization to evs was confirmed by western blot. cell lines stably expressing fluorescent or luminescent reporters were used to assess functional enzyme delivery in recipient reporter cells. results: reporter cells treated with ultracentrifuge pellet material (ucp) from media of transiently transfected cells showed robust and reproducible signal, however fractionating the ucp with an iodixanol density gradient revealed that reporter activity was associated with high-density fractions that were depleted in evs. ucp isolated from identical transfection conditions, but lacking cells (and exosomes), showed identical biological activity levels and distribution in iodixanol gradients, suggesting that the activity was due to contaminating transfection reagent complexes and not evs. serial media changes on ev producing cells post-transfection did not significantly reduce ucp activity on reporter cells. treatment with nucleases did not digest complexed dna, did not significantly reduce dna levels in the ucp as measured by qpcr, and did not decrease activity in reporter cells treated with ucp from either transfected cells or no-cell controls. summary/conclusion: we find that dna-transfection reagent complexes are not separated from evs using differential ultracentrifugation and that common approaches to remove such complexes, including media exchanges and nuclease treatment, are ineffective. due to the pernicious nature of the dna-complex in these cellular assays, it is likely that some reports of ev function are likely artefacts produced by contaminating dna-complexes. we find that density gradient centrifugation can effectively separate evs and dnacomplexes, highlighting the importance of validating elimination of contaminating transfection reagent complexes when using transient transfection to interrogate ev function. chair: suresh mathivanan -la trobe university cancer stem cell-derived exosomes: potential biomarkers for early diagnosis and prognosis in pancreatic cancer introduction: pancreatic cancer (paca) is the most deadly manlignancy, due to late daignosis and early metastatic spread, which prohibits surgery. it is urgently for relaible, early detection. research shows that tumour-derived exosomes, which had been present in the blood in the early stage of tumour formation and before metastasis, is the vanguard forces of tumour formation and metastasis; cancer stem cell-derived exosomes (csc-exos) has stronger migration ability, so the detection of blood csc-exos for early diagnosis and monitoring of progress for paca has great research potential and the value of application. methods: protein markers were selected according to expression in exosomes of paca cell line culture supernatants, but not healthy donors' serum-exosomes. according to these preselections, serum-exosomes were tested by flow cytometry for the pancreatic cancer stem cell marker cd44v6 and tspan8. results: the majority (95%) of patients with paca and patients with nonpa-malignancies reacted with anti-cd44v6 and anti-tspan8. serum-exosomes of healthy donors' and patients with non-malignant diseases were not reactive. recovery was tumour grading and staging independent including early stages. introduction: chronic traumatic encephalopathy (cte) is a tauopathy that affects individuals with a history of mild repetitive brain injury frequently seen in contact sports. initial neuropathologic change of cte include perivascular deposition of phosphorylated tau (p-tau) in cortical neurons and, in later stages, the formation of neurofibrillary tangles in neurons throughout the brain. extracellular vesicles (ev) are known to carry neuropathogenic molecules in neurodegenerative disease and able to cross the blood brain barrier. we therefore examined the protein composition of ev separated from cerebrospinal fluid (csf) and plasma in former national football league (nfl) players with cognitive dysfunction, and an agematched control group with no history of contact sports. methods: evs were separated from csf and plasma from former nfl players (n = 4, 14) and controls (n = 5, 12) by affinity separation method or size exclusion chromatography, respectively. the ev protein profiling was characterized by simoa for tau and ptau and mass spectrometry. the protein data was analysed for ev enrichment, differentially expressed proteins, pathway analysis and correlation with cognitive function, head impact and tau/p-tau levels by biostatistics and bioinformatics. results: the level of total tau and p-tau in csf evs was not significantly changed, but significantly elevated in plasma evs from former nfl players. the 95 proteins were commonly identified between the paired plasma-csf from the same patients, but there was no significant correlation with disease status. collagen alpha-3(vi) chain (col6a3), −1(vi) chain (col6a1) and reelin (reln) were differentially expressed in former nfl players' plasma evs. a combination of these 3 proteins in plasma ev can distinguish former nfl players from controls with 85% accuracy by machine learning. summary/conclusion: the interacting plasma-csf ev proteomes provide an original resource to ev biomarker development for neurodegenerative disease, and col6a3, reln and col6a1 in plasma evs can be potential biomarker for monitoring the cte development. density-based fractionation of urine to unravel the proteome landscape of extracellular vesicles in prostate cancer introduction: current diagnostic tests are unable to discriminate indolent from aggressive prostate cancer (pca), leading to overdiagnosis and overtreatment, and an intense interest in biomarkers to improve clinical decision making. urine is considered an ideal proximal fluid for biomarker identification in pca due to its direct contact with the urogenital system. the discovery and translation of extracellular vesicle (ev) content into pca biomarkers remains challenging due to the difficulty of obtaining urinary ev (uev) with high specificity. methods: we developed a step-by-step protocol to separate uev by orthogonal implementation of ultrafiltration and bottom-up density gradient centrifugation (bu-odg). we implemented complementary particle and protein measurements to identify uev (lower density) and protein rich fractions (higher density) and assess the performance of bu-odg (specificity, efficiency and reproducibility). using mass spectrometry-based proteomics we interrogated uev and protein rich fractions from matched urine and radical prostatectomy tissue samples from pca patients (n = 4), and urine from men with pca prior to (n = 12) and after local treatment (n = 10), benign prostatic hyperplasia (n = 12) and other urological cancers (n = 11). results: bu-odg separated uev from soluble proteins and tamm-horsfall protein (thp) complexes with high specificity and reproducibility, outperforming differential ultracentrifugation, exoquick and size-exclusion chromatography. comparison of the uev proteome from men with benign or malignant prostate disease, allowed us to expand the known human uev proteome and identify a pca specific uev proteome not uncovered by the analysis of the protein rich fraction. proteomic analysis of ev separated from prostate tumour interstitial fluid and matched uev confirmed pca specificity of the uev proteome. analysis of the uev proteome from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uev reflecting their respective cancer tissues of origin. summary/conclusion: we identified hundreds of previously undetected proteins in uev of pca patients and developed a powerful toolbox to map uev and protein rich fractions, ultimately supporting biomarker discovery for urological cancers. immunoglobulin a coating of faeces-derived bacterial vesicles as a marker of inflammatory bowel disease in humans nader kameli a , frank stassen b , heike becker c , john penders c , daisy jonkers d and paul savelkoul b introduction: iga is the most abundant antibody in mucosal secretions and plays a crucial role in maintaining the balance between the host and the gastrointestinal microbiome. recent studies suggested that pronounced iga coating is especially prominent among inflammatory commensals which drive intestinal disease. membrane vesicles (mvs, nano-sized particles released by bacteria) have also been found to interact with the host and modulate development and function of the immune system. however, their interaction with iga has not been studied yet. here we developed a method to isolate and characterize the mvs from faecal samples and checked for possible differences in iga coating patterns of mvs in health and disease. methods: mvs were isolated by using a combination of ultrafiltration and size exclusion chromatography from faecal samples of 6 healthy controls (hc), 6 patients with active crohn disease (cd) and 6 cd patients in a remissive state. quantification and verification have been done with tunable resistive pulse sensing (trpsbased analysis) bead-based flow-cytometer (bbfc) and transmission electron microscope (tem). mvs were selected with specific antibodies for capturing (gram +: lta, gram-: ompa) followed by pe-conjugated anti-human iga antibodies as detection. results: we could successfully isolate 1*109-5*109 particles/ml from 500 mg of faeces. bbfc in combination with trps provide a valuable method for (semi-)quantitative measurements of mixed populations. intriguingly, remarkable differences were found between iga coating mvs derived from healthy controls and active and remissive cd patients as mvs derived from healthy controls were significantly more coated compare to both cd patient groups. in details, for selected g-ve derived mvs: 60% of the total population of mvs derived from hc were coated, 20% from remissive cd patients, and <5% of active cd patients; and for selected g+ ve derived mvs: 55% of the total population of mvs derived from hc were coated, 34% from remissive cd patients, and 25% of active cd patients. (data are represented as the mean). summary/conclusion: here we demonstrate for the first time that mv isolated from the faecal samples are also coated with iga, and surprisingly mvs from healthy volunteers were more densely coated than mvs from diseased patients. the possible consequence of this difference remains to be determined in future studies. monitoring altered tetraspanin and psma expression in prostate cancer derived extracellular vesicles via advanced image flow cytometry (isx) lukas w. prause a , christopher millan b , natalie hensky c , tullio sulser c and daniel eberli c a universityhospital zurich, zurich, switzerland; b university of zurich hospital, schlieren, switzerland; c university of zurich hospital, zurich, switzerland introduction: new diagnostic and therapeutic options for patients with prostate cancer are urgently needed. prostate-specific membrane antigen (psma)-based imaging and therapy are increasingly used for prostate cancer management. unfortunately, as a membrane protein, psma is not found as a soluble protein in the blood and therefore has limited utility as a diagnostic biomarker. however, psma has reportedly been observed as a cargo protein of prostate cancer-derived extracellular vesicles (evs). we demonstrate altered psma expression on evs derived from prostate cancer cell cultures (c4-2, lncap) in response to novel next-generation androgen receptor inhibitor (enzalutamide), a standard chemotherapy agent (docetaxel), a novel experimental nonsteroidal antiandrogen (epi-001) that binds covalently to the n-terminal domain of the androgen receptor and dihydrotestosterone (dht). additionally, evs were isolated from the plasma of prostate cancer patients who participated in the prococ biobank campaign at the usz. plasma was taken and stored from patients both pre-and post-prostatectomy. results: transmission electron microscopy, nanoparticle tracking analysis and simple western (wes) analysis show stable size distribution and amount of evs produced by treated and non-treated cells. using advanced image-based flow cytometry, altered tatraspanin and psma expression could be detected in evs isolated from cell culture supernatants of lncap and c4-2 prostate cancer cells following their treatment. summary/conclusion: measuring psma expression on extracellular vesicles might pave the way to use image flow cytometry of evs to develop a blood based diagnostic test for prostate cancer patients with a wide range of possible applications including: 1) monitoring response to therapy and, 2) early indications of potential relapse. funding: vontobel fondation. proteomic profiling of human neural cells derived extracellular vesicles to identify human brain cell-type specific markers introduction: alzheimer's disease (ad) is a common neurodegenerative brain disease which affects appropriately 30 million patients worldwide. one of the major challenges in ad is to develop reliable biomarkers for early diagnosis and disease-modifying therapies, especially before the clinical symptoms. extracellular vesicles (evs) carry cargos of proteins, lipids and nucleic acids. there was no comprehensive characterization of evs isolated from specific brain cell types, which may be useful for cell type-specific biomarkers. the purpose of this study is to isolate evs from human induced pluripotent stem cell (ipsc)derived brain cells for proteomic profiling and characterization of cell type-specific molecules. methods: human ipscs-derived neurons, microglia and primary cultured astrocytes were differentiated in ev-depleted media. the evs were isolated by differential centrifugation combined with size exclusion chromatography, followed by characterization using nanoparticle tracking analysis and mass spectrometry. the proteomic data were subjected to bioinformatics analysis results: we identified 153 proteins from neuronderived ev (nde), 215 proteins from microgliaderived ev (mde) and 380 proteins from astrocytederived ev (ade) by proteomics. gene ontology analysis indicated that most of these proteins are associated with evs. furthermore, 15, 48 and 251 proteins are present individually in ndes, mdes and ades. among them, high levels of atp1a3 and syt1 in ndes, itgam and cd300a in mdes, and eaat1 and gfap in ades were found, all of which are typically and highly expressed in the original cells. summary/conclusion: our results provide us the potential candidates for cell-type specific ev markers, which will be helpful to develop non-invasive tools to enrich ev originating from specific brain cells and may lead to the development of new biomarkers for neurodegenerative disorders. ) are a tremendous resource for extracellular vesicle (ev) research, but they are heavily focussed on mammalian evs, i.e. evs from humans and laboratory animals, where protein cargoes are well characterised, and a wide selection of antibodies are commercially available. protein markers can be used to identify and define the types of mammalian ev and to determine the presence of any contaminants that might confound functional studies. similar resources are not as readily available for bacterial evs as these are not as well characterised, commercially available antibodies are much less abundant and immunological variation between different bacterial species (and there are 1 trillion bacterial species on planet earth!) means that each species, strain, or group of related species may require different antibodies. methods: to identify quality markers for bacterial evs, we have characterised the proteome of cells, crude evs (ultracentrifuge pellet from cell free culture supernatant) and size exclusion chromatography or density gradient centrifugation purified evs from two different (pathogenic vs probiotic) strains of escherichia coli grown under two different environmental conditions, and one strain of mycobacterium marinum grown in one medium. results: our results identify a selection of proteins enriched in purified ev preparations, and proteins that are depleted after purification steps. summary/conclusion: our results allow the identification of potential markers for ev purity and non-ev contaminants, but also highlight the variability in bacterial ev preparations and suggest potential targets that can be used to investigate the heterogeneity of bacterial ev populations. introduction: recent findings indicate an increase in mid-life mortality rates in the usa and persistent, significant race-related health disparities exemplified by differential mortality rates. this suggests that exploring new molecular markers that may be linked to mortality could provide novel insights into factors that are driving mortality rates. accumulating data suggests that extracellular vesicles (evs) circulating in blood may be potential biomarkers of age-related disease. evs are nano-sized membranous vesicles that bear molecular cargo and mediate intercellular communication between different cells and tissues. little is known about whether ev characteristics differ by race or whether evs are associated with clinically relevant mortality risk factors. methods: in this cross-sectional study, plasma evs were isolated from middle-aged african american (aa) and white males and females. results: we report no significant differences in ev size or concentration with race or sex. there were significantly higher ev levels of phospho-p53, total p53, cleaved caspase 3, erk1/2 and phospho-akt in whites compared to aas. higher ev levels of phospho-igf-1r were found in females compared to males. we examined ev characteristics and protein cargo in the context of well-established clinical mortality risk factors. ev concentration was significantly, and positively, associated with several mortality markers including, high-sensitivity c-reactive protein (hscrp), homoeostatic model assessment of insulin resistance (homa-ir), alkaline phosphatase, pulse pressure, body mass index, and waist circumference. the relationship of ev concentration and cargo with mortality markers differs by race. summary/conclusion: our data show that ev-associated proteins can differ by race and sex and are associated with mortality risk factors. this study provides insight into the characterization of evs in middle-aged aas and whites, which may aid in the development of ev-based diagnostics. funding: this study was supported by the national institute on ageing intramural research program of the national institutes of health. repurposing specialised cell-free dna blood collection tubes for extracellular vesicle isolation introduction: liquid biopsies offer a minimally invasive approach to patient disease diagnosis and monitoring. however, many plasma processing protocols have been designed with a single biomarker in mind. here we investigate whether specialised dna blood stabiliser tubes could be repurposed for the analysis of extracellular vesicles (evs). methods: peripheral blood (n = 3) was collected into k3-edta, roche or streck cell-free dna (cfdna) blood collection tubes and processed using sequential centrifugation immediately or after storage for 3 days. microev were collected from platelet poor plasma by 10,000 g centrifugation and nanoevs isolated using size exclusion chromatography. particle size and counts were assessed by nanoparticle tracking analysis, protein by bca assay and dot blotting for blood cell surface proteins. results: major variations in micro and nanoevs were seen with delayed time to processing. nanoev counts did not change with processing delay or tube collection type but the associated protein amount increased, indicative of cell lysis or activation. the protein was predominantly derived from from platelets (cd61) and red blood cells (cd235a). the increase in associated protein was seen more in the k3-edta and streck tubes indicating that the roche tubes may offer improved cell stability. conversely, microevs increased in both quantity and protein content with delay to processing indicative of both lysis and cell activation, irrespective of tube type. epithelial cell surface marker epcam abundance remained the same across conditions in both micro and nanoevs demonstrating that epcam+ evs were stable. summary/conclusion: specialised cfdna collection tubes can be repurposed for micro and nanoev analysis, however simple counting or using protein quantity as a surrogate of ev number may be confounded by pre-analytical processing. the evs would be suitable for disease selective ev subtype analysis if the molecular target of interest is not present in blood cells. introduction: nutrigenomics and nutrigenetics have been defined as the effect of nutrients on gene expression and genetic variation on dietary response, respectively. here, we propose the isolation and characterization of exosomes from donors carrying different alleles of hla-dqa1 and hla-dqb1, to investigate their involvement in coeliac disease (cd) management. methods: a chilean population (n = 30) was investigated for snps mutations in hla class ii alleles associated to cd predisposition (as well as other mutations related to other food intolerances), using the genochip food technology. exosomes have been isolated from donors' serum by ultracentrifugation and characterized by sds-page, western blotting (cd63 and cd9), and transmission electron microscopy. exosomes were also studied for their interleukins (il-6 and il-1ra) content. results: among the studied population, 86% present at least one of the alleles leading to cd development and 60% carry alleles encoding for αand β-chains heterodimers associated with very high risk to develop cd. in parallel, isolated exosomes from donors with low to extremely high risk for cd showed high il-1ra content (108.8 ± 15.91 to 148.8 ± 12.37), as the persons were not following any treatment. however, values of il-1ra decrease in exosomes isolated form persons receiving treatment for cd. a relationship between exosomes' content and genetic susceptibility for cd has been observed, which may suggest their possible use as biomarkers for cd as the diagnostic of this disease is still a big issue. summary/conclusion: until this point of this underway project, we demonstrate the existence of a relationship between the exosomes' content in il-1ra and genetic susceptibility for cd. furthermore, the genetic predisposition to cd could also modulate the gut colonization process, another important player in intestinal homoeostasis. in the next step, extracellular vesicles from gut microbiota will be isolated and analysed to determine their role in cd management. nasibeh karimi a , razieh dalir fardouei a , jan lötvall a and cecilia lässer b a krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, göteborg, sweden; b krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, gothenburg, sweden introduction: the ability to isolate extracellular vesicles (evs) from blood is vital in the development of evs as disease biomarkers. both serum and plasma can be used but few studies have compared them in terms of amount and type of evs. we have previously developed a method to isolate evs from plasma with minimal contamination of lipoprotein particles (karimi et al 2018) . the aim of this study was to compare the presence of different subpopulations of evs in plasma and serum. methods: blood was collected from healthy subjects, from which plasma and serum were isolated. evs were isolated using a combination of density cushion and size exclusion chromatography (sec) (protocol 1) or a combination of density cushion and density gradient (protocol 2) or immune-capturing (anti-cd63, anti-cd9 and anti-cd81 beads) (protocol 3). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, electron microscopy (em), exoview, flow cytometry and mass spectrometry (lc-ms/ms). results: as determined by nta and protein measurement more evs could be isolated from plasma with protocol 1 and the majority of the vesicles were cd9/ cd41a positive as determined with exoview and western blot. additionally, flow cytometry and western blot showed that more cd9/cd41a positive evs where also identified with protocol 3. furthermore, western blot showed increased amount of cd41a in plasma samples in protocol 2. when labelled evs were spiked in freshly collected blood, no difference in recovery was seen for plasma and serum. summary/conclusion: this study shows that a larger amount of evs could be isolated from plasma compared to serum when three different isolation methods were used. firstly, this suggests that more evs are present in plasma. secondly, it suggests that these vesicles are probably released by platelets and that evs are not trapped in the clot during serum formation. future studies are needed to answer how this affects the use of blood-derived evs as biomarkers from serum and plasma. tumour-derived extracellular vesicles contain distinct integrin proteins stephanie n. hurwitz a and david g. meckes b a university of pennsylvania, philadelphia, usa; b florida state university, tallahassee, usa introduction: cargo profiling, including proteomic analyses, of tumour cell-derived extracellular vesicles (evs) may provide ripe opportunities for further understanding cancer growth, drug resistance, and metastatic behaviour. accumulating data suggest that cancer-derived evs contain membrane-bound integrin proteins which may aid in cell detachment, migration, and homing to future metastatic niches. we have previously published an extensive proteomic profile of secreted vesicles from the nci-60 panel of human cancer cells. methods: here, we further examine the distinct integrin components in these cancer-derived evs, and additionally profile evs released from benign epithelial cells by liquid chromatography and tandem mass spectrometry for comparison. results: we demonstrate the enrichment of integrin receptors in cancer evs compared to vesicles secreted from benign epithelial cells. total ev integrin levels, including the quantity of integrins α6, αv, and β1 correlate with tumour stage across a variety of epithelial cancer cells. in particular, integrin α6 also largely reflects breast and ovarian progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of certain primary tumours. other integrins including α4, αl, and β2 are enriched in vesicles derived from leukaemia cells, and may provide a means to distinguish haematopoietic cell-derived evs. summary/conclusion: this study provides preliminary evidence of the value of vesicle-associated integrin proteins in detecting the presence of cancer cells and prediction of tumour stage. differential expression and selective packaging of integrins into evs may contribute to further understanding the development and progression of tumour growth and metastasis across a variety of cancer types. effect of nicotine and menthol on cytochrome p450 and antioxidant enzymes in rat plasma-derived extracellular vesicles introduction: tobacco products such as e-cigarettes pose potential adverse health effects caused by direct exposure to aerosolized nicotine, flavorants such as menthol, and other particulates. here, we aimed to study the hypothesis that whether nicotine and menthol modulate nicotine-metabolizing cytochrome p450 2a6 (cyp2a6), antioxidant enzymes (aoes), sod1 and catalase in plasma extracellular vesicles (evs). modulation of these enzymes would eventually lead to nicotine-induced toxicity and hiv-1 pathogenesis via evs-based cell-cell interactions. methods: we isolated and characterized evs from rat plasma before and after nicotine self-administration (nic) with audiovisual cue (av) and menthol and characterized using ev markers according to the isev guidelines. protein associated with cyp2a6, sod1, and catalase were quantified by western blot. results: we measured size, total protein, and acetylcholine esterase activity of evs and found no significance difference in these characteristics before and after nic. to investigate the effect av, menthol alone or in combination in the absence and presence of nic, first we evaluated the expression of ev markers cd9 and cd63. the results showed menthol and av together increased the levels of cd9 (p ≤ 0.05), the marker of small vesicles, in the presence of nic. the nic with menthol and av showed a pattern of increased levels of small vesicle but could not reach to significance. next, we demonstrated that the nic with av increased the level of sod1 (p ≤ 0.05), which showed a pattern of increased levels of catalase and cypa6, though statistically non-significant. the expression of nicotine receptor did not change under any conditions used. the results showed an increased level of cyp2a6 (p ≤ 0.01), sod1 (p ≤ 0.05), and catalase (p ≤ 0.05) in plasma evs in the menthol-nic group compared to menthol group only. nic group with a combined av and menthol, showed further increase in the levels of cyp2a6 (p ≤ 0.01), and catalase (p ≤ 0.05). further analysis of plasma evs on inflammatory cytokines/chemokines in these groups, and the effect of plasma evs on nicotine-induced toxicity and hiv pathogenesis are underway. summary/conclusion: nicotine administration increased, though not statistically significant, the levels of circulatory evs. moreover, the study provided evidence that nicotine in the presence of menthol, av, and/or menthol+av increased nicotine-metabolizing cyp2a6 in all the groups and aoes in specific groups. funding: we thank the national institute on drug abuse (grant #da047178, da-047638) for supporting our work. introduction: biomarker discovery in breast cancer (bc) is a clinical need for therapeutics and non-invasive diagnostics. tumour exosomes are involved in premetastatic niche formation and drug resistance and represent a source of non-invasive biomarkers. the identification of tumour exosomal biomarkers provides, not only, the possibility to discriminate patient groups also potential targets to control cancer progression that could be exploited to develop innovate bc therapeutic strategies. methods: we have performed a comparative differenti. al proteomic profile of four bc cell lines and their derived-exosomes, representative of the most relevant bc subtypes in clinic to search non-invasive biomarker candidates. then, we have carried on two bioinformatics approaches: 1) protein association network analysis interaction (string) and 2) pathway inference analysis (hipathia), to characterize the functional profiling for each bc subtype. results: we have found differentially-expressed proteins, in both cells and exosomes, that include indicators of invasion, metastasis, angiogenesis and drug resistance. exosome proteome profile reflects their different bc cell origin suggesting potential indicators of bc subtype. further, bioinformatics analysis reveals a differential role of exosomes in bc signalling pathways in recipient cells, according to their protein cargo and cell origin. summary/conclusion: our results show a set of cells and exosome proteins that highly discriminate bc subtypes and may significantly contribute to further studies for the design of bc biomarker predictor to stratify bc patients and the development of novel therapeutic strategies. funding: a set of potential biomarkers to discriminate breast cancer subtypes. circulatory evs as potential biomarkers of hiv-drug abuse interactions and neurological dysfunction in hiv-infected subjects and alcohol/ tobacco users sunitha kodidela a , kelli gerth a , namita sinha a , asit kumar b , prashant kumar a and santosh kumar a a uthsc, memphis, usa; b university of tennessee health science center, memphis, usa introduction: abuse of alcohol and tobacco can exacerbate hiv pathogenesis and its associated complications. further, the diagnosis of neurocognitive disorders associated with hiv infection and drug abuse using csf or neuroimaging are invasive or expensive methods, respectively. therefore, extracellular vesicles (evs) can serve as reliable non-invasive markers due to their bidirectional transport of cargo from the brain to the systemic circulation. hence, we aimed to study the specific evs proteins, which are altered in both hiv and drug abusers to identify a physiological marker to indicate the immune status and neuronal dysfunction of hiv-positive drug abusers. methods: evs were isolated from plasma of the following subjects: a) healthy b) hiv c) alcohol drinkers d) cigarette smokers e) hiv+alcohol drinkers f) hiv +cigarette smokers. quantitative proteomic profiling of evs was performed by mass spectrometry and potential ev proteins associated with neuronal dysfunction were quantified by westernblot. results: the evs were characterized according to the isev guidelines. a total of 343 proteins were detected in evs of all the study groups. comparison of proteins among all the study groups revealed that hemopexin was significantly altered in hiv+drinkers compared to drinkers and hiv subjects. further, our study is the first to show properdin expression in plasma evs, which was decreased in hiv+smokers and hiv+drinkers compared to hiv patients. though we couldn't identify the few other cns-specific proteins, g-fap and l1-cam, associated with neuronal dysfunction in plasma evs by mass spectrometry, we could detect those by westernblot. the protein expression of gfap (p < 0.01) was significantly enhanced in plasma evs obtained from hiv-positive subjects and drinkers compared to healthy subjects, suggesting enhanced activation of astrocytes in those subjects. the l1cam expression was found to be significantly elevated in smokers (p < 0.05). both gfap and l1cam levels were not further elevated in hiv+smokers compared to hiv+nonsubstance users. summary/conclusion: the present findings suggest that hemopexin, and properdin show potential as markers for hiv-drug abuse interactions. further, astrocytic and neuronal-specific markers (gfap and l1cam) can be packaged in evs and circulate in plasma, which is further elevated in the presence of hiv infection, alcohol, and/or tobacco and thus may represent as potential biomarkers for neurological dysfunction in those subjects. funding: we thank the national institute on drug abuse (da047178) for supporting our work. . electrochemical detection of mirna-21-5p introduction: micrornas (mirnas) are small, single-stranded, non-coding rna species that regulate gene expression post-transcriptionally, and are transported by extracellular vesicles (evs). they play an essential role in biological processes, such as development, cell proliferation, apoptosis, stress response and tumorigenesis. thus, mirnas are considered relevant biomarkers in health. more particularly, mirna-21-5p is expressed in neurons after traumatic brain injury, being expectably transported to peripheral fluids by brain evs that cross the blood-brain barrier. the main goal of this work is to develop an electrochemical biosensor for the detection of mirna-21-5p in serum. methods: overall, the experimental assembly of the biosensor was made in three stages. the first one consisted in the electrodeposition of aunps, the second one in the incubation of anti-mirna21-5p on the carbon screen-s printed electrodes and the final stage in the incubation of mercaptosuccinic acid for blocking unspecific bindings. the probe was hybridized with the target mirna21-5p by a consecutive incubation of several standard solutions. each modification was evaluated with cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis) and square wave voltammetry (swv). the electrochemical behaviour of the biosensor was followed in all steps by monitoring the electron transfer features of a standard redox system. the redox probe selected for this purpose was [fe (cn)6]4-/[fe(cn)6]3-. results: the results indicated that the electrodeposition of gold was more effective for −1.5 v for 600 s and could lead to better signals upon anti-mirna-21-5p hybridization. summary/conclusion: in general, the experiments showed increasing charged transfer resistance upon the incubation of higher concentrations of mirna-21-5p. in these experiments, ev concentration is a critical variable that must be carefully controlled to ensure scientific rigour and reproducibility: without controlling for concentration (dose), experimental outcomes will exhibit excess variability that could mask important biological discoveries. in this study, three orthogonal methods are compared for accuracy in ev quantification: microfluidic resistive pulse sensing (mrps) and nanoparticle tracking analysis (nta) were compared to each other and relative to the gold standard method, transmission electron microscopy (tem). the ability of nta to accurately measure particle concentration is shown to depend on the polydispersity of the sample itself. results validate the accuracy of mrps and emphasize the importance of using orthogonal techniques to quantify evs. methods: reference urinary vesicles were prepared and analysed with the three methods and the relative concentration accuracy of nta and mrps were compared as a function of particle size. the hypothesis that nta concentration accuracy was impeded by sample polydispersity was tested using polystyrene bead mixtures having a range of polydispersity. a theoretical argument based on fundamental physics explains the experimental observations. results: tem and mrps measurements of the evs were in excellent agreement and showed a broad, polydisperse particle size distribution with no peak on the measured size range (50 nm -400 nm diameter). nta differed significantly from tem and mrps by reporting a steep decrease in measured concentration below about 150 nm that resulted in a peak in the reported particle size distribution. bead measurements confirmed the hypothesis to be tested: sample polydispersity significantly affects the ability of the nta method to accurately measure concentration, even for particles as large as 150 nm diameter. summary/conclusion: these experiments validate mrps as an accurate method for quantifying evs and highlight the importance of using orthogonal measurement methods in accordance with misev2018 guidelines. clinically relevant synthetic reference materials to standardize concentration measurements of extracellular vesicles: state-of-the-art and future prospects introduction: there is an unmet need to standardize concentration measurements of extracellular vesicles (evs). flow cytometry is the clinically most applicable method, but the currently available reference materials for calibration are insufficient. for example, the refractive index (ri) between standard particles and evs substantially differs, whereas concentration and fluorescence calibration particles are too bright. the goal of this study is to ascertain the most desired properties of reference materials to standardise ev measurements. methods: an online survey was prepared within the meves ii project to measure the desired size, concentration range, optical properties, choice of fluorochromes, and stability of synthetic ev reference materials for flow cytometry (fcm) measurements. besides the desired properties of ev reference particles, also the available instrumentation was assessed in the survey, which was sent to the members of the stakeholder committee of metves ii project and members of the ev flow cytometry working group. results: the most desired size, concentration, and ri range for ev reference particles is 50 nm to 500 nm, 10e7 to 10e10 1/ml, and 1.35-1.45, respectively. based on mie-theory evaluation of the sensitivity of the available instruments, none of the respondents would be able to detect 50 nm particles with ri = 1.35 with their current instruments. regarding fluorescence intensity, the most desired range according to the responses is from 10 molecules of equivalent soluble fluorochromes (mesf) to 10 000 mesf. considering the sizes of evs and fluorescent labels, the maximal mesf that can be obtained for ev reference particles with 50 nm diameter and high molecular mass fluorescent dyes is in the range of several hundreds. typical antigen densities on evs fall below 100 copies per ev with 50 nm diameter, i.e. mesf values above 100 are probably not physiologically relevant in this size range. summary/conclusion: a part of the desired properties of ev reference materials precludes either their physical feasibility of production or their detection at most currently available fcms, meaning that the intended reference materials will be future-proofed. funding: this work was supported under 18hlt01 metves ii project by the european metrology programme for innovation and research (empir). the empir initiative is co-funded by the european union's horizon 2020 research and innovation programme and the empir participating states. comparison of production and activity of amniotic fluid stem cell extracellular vesicles from 3d hollow fibre bioreactor and 2d culture. culture conditions may affect ev composition and potency. here we compare production, potency, identity and therapeutic potential of evs collected from cells grown in culture dish (2d) versus hfbr (3d). methods: human clonal afsc were derived from patient-consented amniotic fluids. 1x10e6 hafsc were seeded in 2d (145cm2), and 1.6x10e7 hafsc on a small 20kd mwco hfbr (fibercell-c2025d, 450cm2) with fibronectin coating; both cultured in chang medium with 20% of es-fbs, starved for 24 hr and then evs collected. the effect of harvest frequency was tested (8hrs, 24 hr, 72hrs,1 wk). 2d-evs and 3d-evs were compared by nanosight, potency assay (by wb), identity (by exoview analysis) and therapeutic effect (in vivo in an animal model of kidney disease, alport syndrome). results: 2d production was~5.5x10e9 ev/ml/24 hrs while 3d was~2.8x10e10 ev/ml (first four 24 hrs) and 4.4x10e10 ev/ml (two days of hourly harvests). very little difference in ev concentration and very similar size distribution (~130 nm) were observed during harvest intervals; possibly indicating either significant ev re-uptake or inhibition of ev secretion dependent upon free ev in the supernatant. 3d-evs trapped vegf (an in vitro established potency assay) as efficiently as 2d-evs, and expressed cd9, cd81, cd63, cd80, cd86 and vegfr1 as 2d-evs. summary/conclusion: 3d-evs had comparable properties and bio-activity to 2d-evs, but the hfbr produced 10x more evs. hfbr cell culture conditions for hafsc still need optimization, however an available 1.2 m2 cartridge provides a 50x scale up potential. the hfbr, a cgmp closed system, can produce sufficient numbers of ev to support pre-clinical and clinical applications with at least similar properties to evs produced by conventional 2d methods. funding: -intramural funding -intramural ev core pilot funding demonstration of high gain mode in combination with imaging flow cytometry for improved ev analysis luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. in recent years, the importance of evs has become apparent, as they are key mediators of intercellular communication. however, quantifying and characterizing evs in a reproducible and reliable manner is challenging due to their small sizeexosomes range from 30 to 100 nm in diameter. it is well-known that flow cytometers were originally designed to measure and detect cells, and due to the quantitative power flow cytometry offers, there has been a push to quantify and characterize evs using flow cytometric methods. however, these systems have not been designed to measure objects smaller than a cell. methods: here, we describe the use of high gain mode on the amnis® imagestream® imaging flow cytometer to address the challenges of measuring small particles. in this new high gain mode, the charge-coupled device (ccd)-camera is manually adjusted to higher gain settings, increasing the signal obtained from the ev. object thresholds and masking have also been adjusted to better identify and detect small particles. results: preliminary results using murine leukaemia virus-sfgfp reference particles have shown up to a fivefold increase in the number of gfp-positive objects collected in high gain mode, when compared to standard gain on the imagestream system. summary/conclusion: in this study, we demonstrate improved small particle detection, including evs, using this new high gain mode on the imagestream imaging flow cytometer. distance-controlled accelerated catalysed hairpin dna circuit for multiple and sensitive detection of exosomes-associated mirnas introduction: sensitive and simultaneous monitoring of multiplexed exosome-associated rnas is of great value for early cancer diagnosis remains a challenge. methods: here, we report a simple, multiple and sensitive exosomes-associated multiplex mirnas detection method that uses distance-controlled accelerated catalysed hairpin dna circuit (chdc) system without any complex operation or enzymatic amplification. the distance-controlled accelerated chdc can directly enter the plasma exosomes to generate fluorescent signal quantitatively by specifically targeting mirnas without any transfection means. results: we show that distance-controlled accelerated chdc strategy with signal amplification capability could selectively and sensitively identify low level rnas in serum evs, distinguishing patients with early-and late-stage breast cancer from healthy donors and patients with benign breast disease. summary/conclusion: this simple, accurate, sensitive, and cost-effective liquid biopsy by the distance-controlled accelerated chdc method is potent to be developed as a non-invasive breast cancer diagnostic assay for clinical applications. impact of isolation methods on biophysical heterogeneity of single extracellular vesicles university of california los angeles, ca, los angeles, usa introduction: current biophysical analysis of extracellular vesicles (evs) typically encompasses particle density and size distribution determinations using various techniques. however, variabilities in ev isolation methods and the structural complexity of these biological-nanoparticles (sub-100 nm) necessitate more rigorous nanoscale biophysical characterization of single evs to facilitate more reliable and comparable evbased assays. methods: combining atomic force microscopy (afm), super-resolution optical and conventional particle sizing light scatter and microfluidic techniques, we compared the unique sub-nanometre scale biophysical properties of breast cancer cell-derived ev isolates obtained using different isolation methods. results: afm and dstorm particle size distributions showed coherent unimodal and bimodal particle size populations in centrifugation and immune-affinity isolates respectively. more importantly, afm imaging revealed striking differences in nanoscale morphology, surface undulations, and vesicle-to-non-vesicle ratios among ev isolates from different isolation methods. our findings demonstrate the effectiveness of orthogonal high-resolution biophysical characteristics of single evs, not discernable via particle size distributions and counts alone. summary/conclusion: the identified nanoscale biophysical characteristics of ev isolates represent a strategic and complementary framework to resolve differences in the heterogeneity and purity of evs from introduction: extracellular vesicle (ev) concentrations measured by flow cytometry are incomparable. to improve comparability, the metves ii consortium is developing traceable reference materials and procedures, which require validation by test samples. in previous interlaboratory comparison studies, however, a main source of variation was introduced by pre-analytical variables and measurement artefacts introduced by test samples. to minimize variation introduced by test samples, our aim is to develop off-the-shelf biological test samples containing pre-labelled evs. methods: human urine and plasma were collected from healthy donors. evs were labelled with lactadherin-fitc, isolated by size-exclusion chromatography to remove free dye and minimize swarm detection, and mixed with dimethyl sulphoxide (dmso), exocap or trehalose, frozen in liquid nitrogen and stored at −80°c . after thawing, ev concentrations were measured by a calibrated flow cytometer (apogee a60-micro). results: compared to the ev concentrations measured in fresh plasma and urine, the concentrations decreased 27% in plasma (p = 0.04; mean of the 3 cryopreservation agents) and 35% in urine (p = 0.05) after one day of storage. after 5 months of cryopreservation, the concentration of plasma evs decreased 2% (dmso and exocap) and 8.5% (trehalose) compared to one day of storage, whereas the concentration of urine evs decreased 6% (exocap) and 18% (dmso and trehalose). summary/conclusion: we have developed ready-touse, pre-labelled human evs that are stable up to 5 months and dedicated for use in interlaboratory comparison studies. to further increase stability, other cryopreservation agents will be tested. our biological test samples will be key to validate the new reference materials and procedures developed by metves ii in 2021. funding: this project has received funding from the empir program co-financed by the participating states and from the european union's horizon 2020 research and innovation program. understanding intracellular fate of ev-delivered content introduction: despite much work performed on evaluating the potential effects of extracellular vesicles (evs), the functional uptake of their cargo is still controversial. this project aimed to demonstrate that ev content (protein and mrna) is protected and can be subsequently transferred with functional activity into recipient cells, while also developing a tool to assess and quantify functional ev uptake. methods: fusion proteins used were mitochondrial localized coxviii-cfp-nanoluc(cox) and nuclear localized h2b-rfp-nanoluc(h2b). results: hek293 t cell-derived evs protected cox proteins from proteinase k digestion while demonstrating significantly improved efficiency of uptake when compared to free protein, as measured by bioluminescence that was still detectable in recipient cells 96 hrs post-ev-exposure. to confirm functional uptake, recipient cells exposed to evs containing h2b for 72 hrs were imaged and some recipient cells manifested fluorescent red nuclei. to demonstrate the presence of functional mrna within evs, producer cells were transfected for such a duration as not to have detectable levels of protein in the evs while still containing detectable levels of mrna (qpcr) even after rnasea treatment. transfer of these evs to hela cells showed an increase in expression of h2b which was blocked by cyclohexamide, confirming translation of the mrna (2.2kb). to determine if recycling of ev delivered proteins occurs, recipient hela cells were exposed to evs containing cox for 72 hrs. all extracellular evs were removed and cells were trypsinized (0.25% for 30 min) to remove any non-internalized cox protein. 48 hrs later, evs (cd63+ and cd9+) released from cells contained cox suggesting recycling of protein or possibly recycling of entire evs. lastly, an assay was developed to measure functional ev uptake. nanoluc protein was split in two and fused to mturquoise2 (n65) or mscarlet-i(66 c). expression of each fragment alone exhibited non-detectable levels of luminescence while expressing both together had a significantly increased signal. delivery of either fragment within an ev to a cell expressing the corresponding fragment worked as confirmation and quantification of ev uptake (hek293, u87, hela cells). summary/conclusion: this study robustly demonstrates ev delivery of functional mrna and protein to cells, while also establishing a simple assay to quantify and validate functional ev uptake. theoretical model of ev losses due to adsorption on the tube walls. application for immunomagnetic detection of the vesicles introduction: short-term storage of unfrozen samples of vesicles, mainly at 4°c, overnight or during a couple of days is rather common laboratory practice. however, it was found to lead to significant losses of vesicle concentration supposedly due to adsorption on the walls of the tube. the present work develops a theoretical model intended to describe the vesicle adsorption process. the experimental validation of the model was made using method of immunomagnetic precipitation. methods: the theoretical model considers the "diffusion-limited" case of vesicles storage. the maximal adsorption capacity of the surface of contact between the tube and the solution is given as the number of vesicles in hexagonally packed monolayer. for experiment, the vesicles were purified from ht29 cell culture supernatant by differential centrifugation, aliquoted and kept at −80 c. further the aliquots were consequently unfrozen, and placed into the tubes with different surface treatment and kept at +4 c. the kinetics of vesicles loss was measured by anti cd9 immunomagnetic capturing followed by cd81, epcam and cd166 staining and flow cytometry. results: the model allows the estimation of the adsorption-associated losses as dependent on initial vesicles concentration, volume of the solution, tube geometry, the storage temperature and duration case of quiet vesicles storage (without mixing) and also accounts an expected effect of active agitation of the solution (ev-beads complexes formation). theoretical calculations were illustrated by analysis of ev at different storage conditions and during reaction of immunomagnetic precipitation of the vesicles. summary/conclusion: it was demonstrated that application of tubes surface treatment allows increasing sensitivity of immunomagnetic precipitation method to 2x10^5 for cd81+, 5x10^5 for epcam+ and 2x10^8 for cd166+ vesicles. introduction: it is now largely accepted that the intestinal microbiota plays a key role in intestinal bowel diseases (ibd). an imbalance in the composition and diversity of the intestinal microbiota (i.e. dysbiosis) of patients has been repeatedly pointed out by several teams. there are also indications that extracellular vesicles produced by bacteria and exosomes produced by epithelial cells might be increased in this family of diseases. methods: in order to differentiate healthy and ibd faecal samples on the basis of their vesicle profiles, we want to develop a means to enumerate rapidly particles in faecal samples, based on interferometric microscopy. the videodrop technology, developed by myriade, relies on the creation of single beam interferences between two signals from the same light path by nanoparticles such as small vesicles. it will permit to compare on large scales the viral load of healthy subjects and ibd patients. results: this fast and easy-to-use device was compared to the nta on several types of eukaryotic and prokaryotic vesicles and our preliminary results are encouraging. introduction: small extracellular vesicles (sevs) produced by mesenchymal stromal cells (msc-sevs) may be useful in cell-free therapies for immunomodulation and tissue regeneration. methods: to characterize msc-sevs produced ex vivo, human bone marrow mscs were cultured in mesencult-acf plus (macfp), an ev-free and animal component-free culture medium for 3 days and spent medium collected to isolate sevs by ultracentrifugation (uc). analyses of sevs were performed by nanoparticle-tracking analysis (nta), western blot (wb), and human umbilical vein endothelial cell (huvec) tube formation assay. results: analysis of fresh uncultured macfp by uc, nta and wb for cd63, cd81, and cd9 confirmed the absence of sevs. msc-sevs isolated from spent macfp by uc ranged from 80-150 nm in size and were positive for cd63, cd9, and cd81 proteins. these sevs could be stored at −80°c for >4 months in solution or lyophilized with minimal loss based on nta and wb analysis. the msc-sevs contained the msc-associated micrornas let7a, mir21, and mir26a as per qpcr analysis. the biological function of ex vivo isolated msc-sevs was assessed using a human umbilical vein endothelial cell (huvec) tube formation assay. huvecs treated with msc-sevs generated tubes as early as 6 h after seeding, which were not observed in control huvec cultures until 15 h. moreover, the number of branch points present in such tube structures was >fourfold higher in huvec cultures (n = 5) supplemented with msc-sevs versus control, with the former lasting >60 h and the latter lasting <50 h in culture. direct comparison of the performance of macfp medium to media containing non-depleted or ev-depleted foetal bovine serum demonstrated that only mscs cultured in macfp (n = 3) were able to expand robustly with a doubling time of 1.1, 2.1 and 8.9 days in these media, respectively. lastly, methods for isolating sevs using newly developed easysep-ev™ magnetic separation kits and size exclusion columns will be presented. summary/conclusion: taken together, these data demonstrate that msc-sevs can be produced in high yield in macfp medium and that these possess similar physical, phenotypic and functional characteristics as sevs in vivo. funding: this work was privately funded by stemcell technologies inc. introduction: extracellular vesicles (evs) are heterogeneous group of small vesicular structures released by different types of cells, including stem cells (scs). as recent studies demonstrate that they may enclose bioactive content and transfer it into the target cells, growing interest is placed on the utilization of evs in the field of biomedical research. however, there is still lack of standardized methods of evs characterization. as an example, typical flow cytometry-based protocols, commonly used for cells phenotyping, may be inadequate for the characterization of evs as particles with size close to the detection limit of conventional cytometers. thus, the aim of this study was to optimize and compare the use of different flow cytometry platforms for the multiparameter analysis of evs isolated from different types of scs populations. methods: ev samples were obtained by ultracentrifugation of conditioned media collected from selected scs types, including human induced pluripotent scs (ips) and mesenchymal scs (mscs). next, several high resolution flow cytometry systems: cytoflex, apogee (a50 and a60 micro-plus) and image stream mk ii were employed to compare their sensitivity and resolution, as well as influence of "swarm" effect. furthermore, we examined evs phenotype, including expression of tetraspanins and other surface markers. results: our results have revealed that tested flow cytometry systems may be utilized for the phenotypic characterization of evs secreted by scs populations. however, the conventional staining and gating strategy protocols have to be thoroughly optimized. additionally, depending on a type of tested cytometer, we have demonstrated the difference in a "swarm" effect and its influence on obtained results regarding evs phenotype. finally, imaging flow cytometry platform was also employed to visualize evs on the single particle level. summary/conclusion: in conclusion, we have demonstrated that tested high-resolution flow cytometry platforms are convenient methods for the multiparameter characterization of evs produced by different types of scs populations. however, careful selection of particular measurement parameters should be performed depending on a type of employed system. funding: this study was funded by ncbr grant strategmed iii (strategmed3/303570/7/ ncbr/2017) to ezs. evaluation of atcc's exosomes from cell culture supernatant as reference standards in research and development. introduction: exosomes are subcellular particles 30-150 nm in size released from cells through a fusion of multicellular bodies with the plasma membrane. exosomes are stable carriers of cell-free cargo in the form of dna, rna, and proteins, thereby making them an attractive candidate for diagnostic and therapeutic applications. however, isolating a consistent population of exosomes can be challenging and there is an unmet need for highly characterized exosomes for use as reference standards in extracellular vesicle research (ev). methods: exosomes were isolated from cell culture supernatants of different atcc cell lines including stem cells and cancer cell lines representing the most prevalent cancer types -prostate, colorectal, breast, lung, cervical and glioblastoma, using tangential flow filtration (tff). these exosomes underwent sterility and mycoplasma tests as a part of their quality control. the morphology and size distribution of these exosomes were evaluated through multiple strategies including nanoparticle tracking analysis (nta), asymmetrical flow field-flow fractionation (af4), cryo-electron microscopy (cryo-em) and spectra dynetm particle analyser. exosome surface markers were also analysed through multiple strategies such as electro chemiluminescent elisa, flow cytometry and western blotting. also, stem cell exosomes and cancer exosomes were further evaluated for functionality through in vitro functional assays including migration assay, angiogenesis and anchorage independent growth assay. results: our optimized tff method resulted in high yields of > 1 × 1010 exosomes/ml and average protein equivalent of more than 1 mg/ml. more than 90% of the exosomes population had an average size distribution of 50-200 nm and median size of 110 nm confirmed through a number of different size distribution instruments. although cell line dependent, we were able to obtain similar expression levels of different cell surface markers including tetraspanins (cd63, cd81, cd9) when evaluated through different methods. our functional data demonstrated stem cell exosomes were functionally active in promoting cell migration and tubule formation. additionally, cancer cell exosomes were found to promote a malignant phenotype in an anchorage independent growth assay. summary/conclusion: collectively, we demonstrated our ability to reproducibly manufacture production-scale batches of exosomes from multiple different cell types. our purified exosomes are of high yield, meet well-established quality control specifications, and are robust in maintaining size distribution, surface marker expression, and functionality in vitro. therefore, they can serve as ideal reference materials that can support different ev-based research applications. exo-cise: extracellular vesicles enriched from plasma post-exercise promotes myogenesis and neurogenesis bianca paris a , yaomeng liu a , vicente pagalday-vergara a , julie davies b , priya samuel a , ayman abu seer a , johnny collett a , laura gathercole a , ken howells a , karl j. morten b , zhidao xia a , daniel anthony b , david r f. carter a , helen dawes a and ryan c. pink a a oxford brookes university, oxford, uk; b university of oxford, oxford, uk introduction: physical activity brings about a widespread physiological response and elicits the beneficial adaptation of several tissues and organs. furthermore, regular participation in physical activity reduces the risk of developing major non-communicable diseases such as cardiovascular disease, diabetes, cancer, osteoporosis, and dementia. two important processes known to occur following physical activity are myogenesis and neurogenesis; both of which involve the activation and proliferation of specialised tissue-resident stem cells. the molecular mechanisms regulating these processes following exercise are poorly understood to date. here, we investigated the contribution of extracellular vesicles, which are released into the circulation after exercise, to benefit adult myogenesis and neurogenesis. methods: small extracellular vesicles were enriched from the blood of healthy participants before and following maximum and moderate intensity exercise. differentiation and proliferation using a range of methods was measured following vesicle treatment onto primary myoblasts and neuronal primary exvivo stem cells. activation of key cellular pathways were measured. results: we show significant proliferation and differentiation changes of both stem cell types. this is independent of extraction method, extracellular vesicle depleted fractions and is interestingly conserved across mammalian species. remarkably, we see an age-related effect. summary/conclusion: this advocates that short single bouts of exercise may promote myogenesis and neurogenesis via systemic signalling of extracellular vesicles which opens an interesting field in endogenous ev therapies. show promise as a cell-based therapy for retinal degeneration. while clinical trials are ongoing, the potential of extracellular vesicles (evs) as biomarkers for monitoring eye health and disease is not well studied. this study characterized the ev surface profile and cargo of hipsc-rpe to offer a baseline assessment in normal and disease conditions. moreover, we evaluated the importance of pnpla6, a gene involved in membrane integrity and when mutated causes retinal degeneration, in ev biogenesis and secretion. methods: evs were isolated from serum-free culture medium of hips-rpe and identified with nanoparticle tracking analysis, transmission electron microscopy, and immunoblot analysis of exosomal markers, including alix, tsg101, and cd63. surface marker detection and proteomic profiling were completed using an ev surface marker kit and mass spectrometry, respectively. small interfering rna targeting pnpla6 was used to knockdown the expression in hipsc-rpe and evs were characterized. results: nanoparticle tracking analysis confirmed the presence of both microvesicles (>150 nm) and exosomes (<150 nm) by size distribution and the concentration of evs (1x108 particles/ml) from rpe. tem displayed typical morphological characteristics of evs. the presence of known ev markers, alix, tsg101, and cd63 was confirmed via immunoblot and flow cytometry. surveillance of ev surface markers revealed enrichment of epithelial markers (cd326) and stem cell markers (cd133/1) that depict donor cell origin and functional proteins including integrin-binding (cd29) and tgf-beta receptors (cd105). in addition, proteomic analysis revealed regulators of inflammation and rpe function, including hemopexin, clusterin, complement factor i, and pigment epithelium-derived factor. furthermore, reduction in pnpla6 expression reduced vesicle secretion and vesicle size compared to non-targeting controls. introduction: vascular endothelial growth factor (vegf) is a potent angiogenic factor and was first described as an essential growth factor for vascular endothelial cells. vegf plays a role in normal physiological functions such as bone formation, haematopoiesis, wound healing, and development. mesenchymal stem cell (msc) was found to secretes potential growth factors such as vegf when cultured in vitro. however there are some beliefs that foetal bovine serum (fbs) which usually used as serum in cell culture content vegf. methods: msc seeded in in 96-well plate in with concentration of 5,000 cell/well. cells were incubated for 24 hours and fasted for another 16 hours using only dmem. cells were treated with complete medium consist of dmem and 10% fbs. culture medium were collected after 5, 12, and 24 hours after treatment. cell were culture in 37ºc dan 5% co2. vegf concentration was detected using elisa technique. results: vegf concentration was not found in fbs which do not contact with msc. an increasing of vegf concentration in time-dependent manner was shown when culture medium was used in msc cell culture in normoxic condition. the result of vegf concentration when culture 5, 12, and 24 hours were 74.022 pg/ml, 76.67 pg/ml, and 93.58 pg/ml, respectively. the mechanism of msc release growth factor is still under investigated. however, the classic growth factors and cytokines serves paracrine control molecules which were important in regenerative medicine. vegf was found to be an important molecules in angiogenesis process and determine the fate of cells. summary/conclusion: msc secreted vegf and concentration increased in time-dependent manner. isolation and characterization of exosomes from canine stem cells introduction: unlike induced disease models using laboratory animals, naturally occurring disease models display pathophysiologic attributes that are more similar to human diseases. unfortunately these models are underutilized in translational regenerative medicine research. this is partly due to the slow development of species-specific experimental therapeutics to investigate comparative efficacy. thus, we set out to isolate and characterize exosomes from canine adipose-derived mesenchymal stem cells (cad-msc) to use as a comparative therapeutic in dogs. to accomplish this, we optimized an isolation and purification strategy and characterized their molecular properties. methods: exosomes were isolated by sequential centrifugation and subsequent ultrafiltration. the proteome was characterized by tandem mass tag (tmt) mass spectrometry and the mirna cargo was identified using a canine specific pcr array with subsequent target and enrichment analysis using targetscan and the panther platform, respectively. also, nanoparticle tracking analysis and transmission electron microscopy were used to determine exosome size and structure. to investigate bioactivity, we measured the ability of exosomes to inhibit collagen production in an in vitro model of fibrosis. results: exosomes were purified by ultrafiltration using a 100 kda cut-off. proteomic analysis by tmt mass spectrometry identified 1262 unique proteins. 90% of the exocarta top 100 were identified from this list. additionally, we identified the mirna cargo within exosomes and found 27 highly expressed mirnas. enrichment analysis identified multiple pathways of probable regulation including angiogenesis (fold enrichment = 3.1; p < 0.0001) and transforming growth factor-beta (tgfb) signalling (fold enrichment = 4.1; p < 0.0001). exosome size was quantified to be 119.7 ± 3.4 nm with a modal average of 98 nm. lastly, in the presence of exosomes, tgfb stimulated fibroblasts deposited 32.2% less collagen than vehicle controls (p = 0.036). summary/conclusion: in summary, cad-mscs exosomes display structural and functional features comparable to stem cell derived exosomes from other species. use of these exosomes in naturally occurring disease canine models may provide superior predictive value for human clinical trials. funding: support provided by the ccah, school of veterinary medicine, uc davis. mesenchymal stem cells-derived exosomes promote in vitro the progression of triple negative breast cancer cells introduction: mesenchymal stem cells (mscs) are multipotent stromal cells and have been described as key regulators of different aspects of tumour physiology. in tumour pathogenesis, mscs can integrate the tumour microenvironment after recruitment and are able to interact with cancer cells to promote tumour modifications by affecting epithelial-tomesenchymal transition (emt). it was revealed that exosomes derived from mscs are critical players in the tumour niche. exosomes are a novel way of cellto-cell communication and play crucial roles in the majority of pathways that contribute and affect response to therapy, cell-adhesion molecules and the progression of tumour cells. because of the known importance of this communication we decided to investigate the implication of mscs with triple negative breast cancer (tnbc) cell lines as well as exosomal profiles between the experimental conditions. methods: the interactions of mscs with triple negative breast cancer cell lines (mda-mb-231 and hs578t) was performed by coculturing mscs (or tnbc cell lines) with exosomes derived from tnbc cell lines (or mscs). physical characterization of isolated exosomes was performed followed by their molecular investigations. cell proliferation was detected by mtt assay and migration was analysed by wound healing assay using 2d cultures. moreover, we also used 3d culture to assess the exosomes uptake and to observe their capability of internalization into a 3d structure. the alterations in expression level of some transcripts (mrnas and mirnas) and protein profile were investigated by qrt-pcr, western blot and immunofluorescence staining. results: we found that mscs-derived exosomes are actively incorporated by triple negative breast cancer cell lines (2d culture). in coculture, in tnbc cells the expression level of mesenchymal markers and emt markers (e-cadherin, vimentin) at mrna and at protein levels, as well as mirna-derived exosomes targeting mesenchymal genes were significantly affected. using bioinformatics tools, we highlighted the important biological processes which were activated by promoting tumour modifications. in addition, using 3d culture we provided a comprehensive understanding regarding exosomes internalization in 3d structures, which closely mimics in vivo conditions, compared to 2d culture. summary/conclusion: in this work, we focus on the investigation of mscs-derived exosomes in order to highlight their implication in several biological processes, including tumour proliferation and progression of triple negative breast cancer cells. all these alterations affect the response to therapy and should be considered for developing efficient therapeutic strategies. natural killer cell-derived extracellular vesicles have a potent anti-leukaemic effect and selectively target the cancer stem cell subpopulation introduction: natural killer (nk) cells of the immune system recognize and kill tumour cells. extracellular vesicles (evs) secreted from nk cells are capable of killing tumour cells independent of the cell to cell contact required for nk cell activation. cancer is a leading cause of death, primarily due to metastasis and recurrence. cancer stem cells (csc) within tumours are resistant to chemotherapy and immune attack, and cause metastasis and relapse. identification of the cancer types killed by nk evs is limited, and the effect of nk evs on cscs has not been described. here we determine whether nk-derived evs kill a myeloid leukaemia cell line and its csc subpopulation. methods: nk evs were isolated from our nk cell line, nk3.3, derived from normal human lymphocytes. nk3.3 evs were characterized by immunoblotting, proteomics, and next generation rna sequencing. human k562 leukaemia cells were treated with nk3.3 evs in vitro and analysed for proliferation and markers of cell death. results: nk3.3 evs contain ev-associated proteins alix, cd63, hsp70, and tsg101, nk effector molecules perforin, granzymes a and b, granulysin and nklam/ rnf19b, an e3 ubiquitin ligase required for maximal nk cytotoxicity, and tumour suppressor mir-186. nk3.3 ev treatment of k562 significantly decreased its expression of proliferation markers cd71 and ki67, and increased the frequency of apoptotic and necrotic cells, paralleled by elevated levels of active caspases −3 and −7. non-tumorigenic cells were unaffected by nk ev treatment. most notably, nk3.3 ev treatment significantly reduced the frequency of k562 cells highly expressing aldh, a csc marker. summary/conclusion: nk3.3-derived evs have a robust anti-tumour effect on k562 myeloid leukaemia cells and selectively target the csc population, suggesting they may circumvent the evasion and resistance mechanisms used by cscs. nk3.3 evs therefore have introduction: due to their potential as a key bioactive agent in regenerative medicine applications, mscderived extracellular vesicles (msc-evs) are increasingly being investigated as a clinical therapy. manufacturing that generates enough evs for product development and clinical doses is currently a limitation in the field and clearly a scalable manufacturing solution will be necessary for successful translation. moreover, a complementary approach that increases the ev productivity, i.e. the number of evs produced per cell, could further help to accelerate the development of msc-evs as a therapy. methods: we developed a process that leverages a series of new cell culture reagents to couple to our established cell-media system for scalable manufacturing of msc-evs. briefly, human bone marrow-or umbilical cord-derived mscs were rapidly expanded under xeno-free conditions (i.e. >150x expansion within 10 days). cultures were then switched to our proprietary ev collection medium and evs were harvested for up to three additional days. at the end of culture, the evs in the conditioned media were concentrated using a tangential flow filtration (tff) system. to increase the productivity of mscs, two medium supplements were developed that increased ev yield by either increasing the number of evs generated per cell in a shortened culture process or increasing the number of collected evs by lengthening the ev collection culture period. results: this scalable msc-ev manufacturing method was implemented in both 2d flask and 3d bioreactor culture and generated over 2,000 particles per cell in 2d and over 4,000 particles per cell in 3d. with the addition of a medium supplement to increase evs produced per cell, the ev productivity was increased >2x after 24 hrs. alternatively, ev productivity was also increased >2x by addition of the medium supplement that extended ev collection culture period. summary/conclusion: msc-ev success in clinical translation will be reliant on a manufacturing method that can scalably and reliably generate large amounts of evs. these results present one such solution. furthermore, increasing ev productivity, for instance by medium supplements that increase evs per cell or lengthen culture times could further address the limitation of generating the evs required for development and translation of clinical therapies. simplifying scalable msc ev production in a microcarrier-based bioreactor system divya patel, josephine lembong, katrina adlerz, jon rowley and taby ahsan roosterbio inc, frederick, usa introduction: the growpt0ing numbers of msc-ev clinical applications drives the need for a scalable msc-ev production platform. while most msc-evs are generated while cells are attached to tissue culture plastic, such 2d cultures cannot be scaled up to meet the yields necessary for commercialization of ev-based therapeutics. we have shown that 3d bioreactors can be used to generate msc-evs and that paradigm can be scaled directly in terms of yield from the 3 to 15 l scales. the technical expertise of seeding cells onto microcarriers for expansion in bioreactors, however, requires technical expertise not available to all those in the ev field. therefore, our goal here is to simplify and expedite the ev collection process in bioreactors by cryopreserving cells on microcarriers, such that end users can merely thaw and then collect msc-evs. methods: mscs were expanded in 2d and then seeded on three different microcarriers and cultured in a bioreactor for 5 days. when confluent, cells on microcarriers were cryopreserved. to evaluate the microcarriers and the cryopreservation protocol, the cells-microcarriers were thawed, cultured in a bioreactor in growth media for 24 hours, then in ev collection media for 3 additional days. cell recovery and ev production upon thaw was evaluated and compared to ev collection from fresh, non-cryopreserved cells. results: total cell counts 24 hrs post thaw were comparable to those before cryopreservation and to fresh samples prior to ev collection. following 3-day ev collection, concentration of particles collected from cryopreserved cells on microcarriers were similar to those collected from the fresh cells (2e9 particles/ml). this process was validated for two different microcarriers using two separate cryopreservation solutions. summary/conclusion: our results show that cryopreserved hmscs on microcarriers can support ev collection in a 3d bioreactor process with a particle yield that is comparable to those collected from fresh cells. this cryopreserved product can simplify ev production, reducing cost and time by removing process steps associated with the hmsc expansion, with in a paradigm suitable for scale-up. the whitening, anti-wrinkle, and wound-healing effects of extracellular vesicles from orbicularis oculi muscle-derived stem cells. introduction: skeletal muscle-derived stem cells possess potent therapeutic activities in the treatment of muscle-related disorders. in our study, we tried to isolate and characterize orbicularis oculi muscle (orm)-derived stem cells (orm-scs) from the discarded human tissues which were obtained from the ocular surgery-subjected patients. we also prepared the natural extracellular vesicles (evs) from the cultured orm-scs and assessed the their therapeutic actitities including the skin whitening, anti-wrinkle, and wound healing effects. methods: we isolated the orm-scs from the patients subjected to ocular surgery and characterized the orm-scs by analysing cell morphology, proliferation, expression levels of the cell surface and stemness-associated markers, and tri-lineage differentiation and colony-forming capacities, confirming the stemness properties of the orm-scs. then, we prepared the natural evs from the orm-scs via the centrifugation and filtration of the media supernatants and their therapeutic activity was investigated. results: the isolated orm-scs showed spindle-like morphology and positive expression of cd105, cd 90, and cd73, but they were negative in expression of cd45 and cd34. the orm-scs showed the capacity of osteogenic, adipogenic, and chondrogenic differentiations. the evs from orm-scs (orm-sc-evs) possessed the apparent inhibitory effect on the melanin synthesis in b16f10 cells by blocking the tyrosinase activity, although orm-sc-evs treatment did not dramatically change the expression level of melanogenesisrelated genes, such as microphthalmia-associated transcription factors (mitf), tyrosinase (tyr), tyrosinaserelated protein1(tyrp-1), and tyrp-2. in addition, we confirmed that orm-sc-evs could stimulate skin cell migration and increase the expression level of antiwrinkle related genes and wound-healing properties. summary/conclusion: this study revealed the stem cell property of orm-scs and the whitening, antiwrinkle, and wound healing effects of orm-sc-evs, suggesting that orm-scs and orm-sc-evs can be successfully used for stem cell-based ev therapy and cosmetics, by regulation the melanogenesis, wrinkle, and wound. funding: this work was supported by grants from the national research foundation (nrf) funded by the korean government (2019m3a9h1030682). use of stem cell extracellular vesicles as a holistic approach towards cns repair introduction: neurological diseases and disorders are leading causes of death and disability worldwide. many of these pathologies are associated with high levels of neuroinflammation and irreparable tissue damage. we have previously shown that extracellular vesicles (evs) from infected cells contain viral by products (noncoding rnas and proteins) and that these evs can exert deleterious effects on recipient cells1-3. therefore, in the context of neurotrophic viruses evs may contribute to or perpetuate processes relating to neuroinflammation and neurodegeneration. due to their multipotent properties, stem cells have broad applications for tissue repair; additionally, stem cells have been shown to possess both immunomodulatory and neuroprotective properties. in recent years it has been well-established that stem cell evs play a critical role in the functionality associated with stem cells. the diverse biological cargo contained within these vesicles are proposed to mediate their effects and, to date, the reparative and regenerative effects of stem cell evs have been demonstrated in a wide range of cell types. while a high potential for their therapeutic use exists, there is a gap of knowledge surrounding their characterization, mechanisms of action, and how they may regulate cells of the central nervous system (cns). methods: we have isolated and recovered high yields of evs from large scale cultures of both induced pluripotent stem cells (ipscs) and mesenchymal stem cells (mscs) using tangential flow filtration. our ev characterization includes both phenotypic (size, tetraspanin expression) and biochemical assays. ev functionality has also been assessed in vitro utilizing several cellbased assays related to cellular viability, migration, angiogenesis, and immunomodulation in both healthy and damaged recipient cells with relevance to the cns. results: our data suggests that evs from different sources of stem cells display unique phenotypes, exhibit differential association with various cytokines, proteins, and long non-coding rnas, and have the ability to significantly enhance processes that are critical for cellular repair4. lastly, utilizing an ipsc-derived neurosphere model, we have observed a robust uptake of stem cell evs and have found that these evs are able to effectively penetrate these 3d structures. summary/conclusion: collectively, these results highlight the "holistic" properties of stem cell evs by demonstrating their ability to partially reverse or reduce damage in various cell types. funding: this work was supported by national institutes of health (nih) grants ai078859, ai074410, ai127351-01, ai043894, and ns099029 to fk and r33 ca206937 and r01ar068436 to lal. the effect of cell culture media on extracellular vesicle secretion from mesenchymal stem cells and human pluripotent stem cell-derived neurons introduction: cell culture media and its supplements are known to affect the secretion and isolation of extracellular vesicles (evs) from cell cultures. identification of these effects is crucial especially when planning to use evs as therapeutic agents. here, we investigated the effect of cell culture media on ev yield from human mesenchymal stem cells (mscs) and human pluripotent stem cell (hpsc)derived neurons. methods: evs were collected from cell-conditioned media (ccm) and no cell control (ncc) media using size-exclusion chromatography (sec). mscs were cultured in dmem/f12:neurobasal medium or in opti-mem reduced serum medium, both supplemented with exosome-depleted foetal bovine serum (fbs). the ev yield from hpsc-derived neurons was compared at two maturation time points (day 46 and 60), in dmem/f12:neurobasal or in opti-mem, with and without 3-hour kcl stimulation. sec fractions were analysed by nanoparticle tracking analysis (nta), protein concentration assay and blinded transmission electron microscopy (tem). results: ccm samples had a clear peak of evs in sec fractions 7-10, which was not detected with ncc. interestingly, a second population of evs eluted in sec fractions 13-17 in both ccm and ncc, indicating presence of evs in exosome-depleted fbs. moreover, this second population differed largely between used media batches. culture medium had no significant effect on msc ev yield (dmem: 8.30e+08 particles/ ml, opti-mem: 9.61e+08 particles/ml). with neuronal cultures, no significant differences in ev yield were found between culture media or cell maturation time points. in contrast to earlier findings, 3-hour stimulation of neurons by kcl resulted in significantly smaller ev yield compared to non-stimulated controls (stimulated: 5.28e+10 particles/ml, non-stimulated: 1.43e+11 particles/ml, p < 0.02). summary/conclusion: our results indicate that exosome depleted-media are not entirely devoid of vesicles, which can cause bias in downstream analyses. however, sec is a good method to separate cellsecreted evs from the contaminating medium-derived evs. culture medium did not affect the number of evs secreted by mscs or neurons; instead, we observed larger differences between media batches. this data emphasizes the importance of analysing the ncc as negative control in all cell culture experiments. mouse mesoangioblast stem cell extracellular vesicles are able to influence macrophage cell activity maria magdalena barreca a and fabiana geraci b a dept stebicef university of palermo, palermo, italy, palermo, italy; b dept stebicef, university of palermo, italy, palermo, italy introduction: it is largely demonstrated that stem cells release extracellular vesicles (evs) that are able to modify target cell behaviour. interestingly, there is a bidirectional signalling exchange between stem cell evs and damaged cells. moreover, it is well known that macrophages, could also play a role in wound repair and tissue regeneration. it was also demonstrated that stem cell evs are involved in immune cell regulation. for this reason, today takes hold the idea that evs could replace stem cells in regenerative medicine. the aim of our work was to evaluate if evs released by mouse mesoangioblast stem cells (a6) could have a role in immune cell regulation. specifically, we have investigated the possible a6 ev effect on murine macrophages (raw264.7) in terms of cell proliferation, migration and phagocytic ability, and cytokines/chemokine release. methods: a6 evs were collected from conditioned milieu by ultracentrifugation. raw 264.7 cell proliferation with or without a6 evs was evaluated via cfse assay. scratch test was performed to assay their migration ability. to study raw264.7 cell phagocytosis they were treated with 2 μm beads. finally, cytokine array was used to monitor their secretion after ev treatment. results: we have found that a6 evs inhibited macrophage proliferation as proved by a proliferation index significantly reduced after ev treatment. simultaneously, we have noticed that evs increases raw264.7 migration ability. furthermore, a6 evs are able to increase macrophage phagocytic activity. as it is known that hsp70 is involved in for macrophagic activity increase and a6 evs express hsp70 on their surface, we performed phagocytosis assays assay by blocking the protein or its receptor tlr2, tlr4 and cd91. our data demonstrated that a6 evs increase phagocytosis through hsp70 and its receptors. we have also proved that a6 evs modify the expression pattern of cytokines/chemokines released in the extracellular milieu by raw264.7 cells. in particular, we observed an increase in anti inflammatory cytokines, and a decrease in some inflammatory ones, suggesting that evs could polarize macrophages towards an anti inflammatory m2 phenotype. summary/conclusion: in conclusions, our data show that a6 evs influence macrophage activity and additional studies could provide a new insight into understanding the underlying potential of evs in tissue regeneration. 1 and 2) . in a healthy kidney, the polycystins localize to renal cilia. mutations that abrogate ciliary localization of pkd2 (yet preserve its channel function) also cause cysts. besides cilia, pkd2 is also found in other subcellular locations including extracellular vesicles (evs) of human urine. how dysfunction of pkd2 trafficking and localization leads to the kidney pathology remains unknown. pkd2 is evolutionarily conserved across all members of eumetazoa. in c. elegans, pkd-2 is exclusively expressed in ciliated male-specific neurons, where it is trafficked to cilia and evs. gfp-tagged pkd-2-containing evs play a signalling role in inter-organismal communication between animals. conservation of polycystin-2 cellular localization between worm and human suggests that their network of molecular interactions may also be conserved. we propose that pkd-2 plays distinct roles in cilia versus ciliary evs. methods: to understand the role of evs in c. elegans inter-organismal signalling, we aim to identify the pkd-2-associated ev proteome, transcriptome, and metabolome. we established a pipeline for fluorescent labelling and tracking specific ev cargoes in a living animal using super-resolution microscopy. we used fluorescence of the pkd-2 carrying evs to optimize biochemical procedures for their enrichment. results: our initial analysis revealed two populations of pkd-2-carrying evs that differ in their densities: 1.11-1.12 versus 1.14 g/ml. we are currently characterizing these two distinct populations using transmission electron microscopy and refining our enrichment procedure for protein identification by mass spectrometry, sequencing of their rna cargoes and metabolome analysis. summary/conclusion: what function human pkd2 plays within the cilia and within the urinary evs is not well understood. identification of molecular mediators of c. elegans pkd-2 ev signalling will inform on the interactome of human pkd2 and its function in cilia versus evs. introduction: ectosomes play roles in many physiological and pathophysiological processes, and their precise is dependent on molecular cargo and parent cell type. a single cell can release distinct subpopulations of evs enriched with different molecular cargo, which adds complexity to elucidating cargo sorting and biogenesis mechanisms. in the nematode c. elegans, ectosomes bud from sensory neuron cilia and are released into the environment to modulate animal behaviour. methods: c. elegans is genetically tractable and optically transparent, allowing for live imaging of fluorescently tagged ev cargo. we express all tagged cargo at endogenous levels, adding physiological relevancy. results: we discovered that the calcium homoeostasis modulator ion channel clhm-1 localizes to cilia of ev-releasing neurons and observed gfp-tagged clhm-1 in ciliary evs. using super resolution microscopy, we imaged evs released from animals coexpressing tdtomato-tagged clhm-1 and gfp-tagged pkd-2 (another vesicle cargo) in the same neurons. while the two proteins colocalize in the cilia, clhm-1::tdtomato and pkd-2::gfp rarely colocalize in evs. this indicates that separate subpopulations of evs are being released from the same neurons. to determine how the clhm-1 subpopulation is formed, we are investigating candidate genes. anoh-1, a homolog of the ca2+ scramblase tmem16f, localizes to neuron cilia and induces phosphatidylserine exposure on the outer membrane leaflet. in anoh-1 mutants, the number of clhm-1::gfp evs released is significantly decreased but the number of pkd-2::gfp evs does not significantly change. in addition, i am using facs to isolate clhm-1 and pkd-2 containing evs and analysing the respective proteomes with lc-ms/ms. summary/conclusion: we are elucidating mechanisms that give rise to distinct subpopulations of ciliary evs in c. elegans and defining cargoes being enriched in these ev subpopulations to gain insight into ev cargo sorting and biogenesis mechanisms in ciliated neurons. ceramide accumulation induces exosome secretion through lysosomal protein laptm4b kohei yuyama, hui sun and yasuyuki igarashi hokkaido university, sapporo, japan introduction: exosomes, a type of extracellular vesicles originated from multivesicular bodies (mvb), are important carriers of cellular molecules and have critical roles in intracellular communication in both health and disease. ceramides (cer) are implicated in biogenesis of exosome, however the molecular machinery that mediates exosome secretion remains obscure. lysosome-associated protein transmembrane-4b (laptm4b) is a lysosome/late endosome-resident transmembrane protein, which has been reported to bind cer. we demonstrate here that laptm4b is involved in the exosome secretion, which are induced by exogenous cer treatment or lysosomal ceramidase inhibition in cultured neuronal cells. methods: neuroblastoma sh-sy5y cells were treated with cer (porcine brain-derived cer or synthetic d18:1/c2:0~c24:0 cer) for 24 h. exosomes were isolated from the culture supernatants by sequential centrifugation and their amounts were measured using ps capture exosome elisa kit. to analyse mvb transport, mvb and recycling endosomes are visualized with gfp-cd63 and rab11 immunostaining, respectively. results: we found that exogenous treatment of cer, especially those with c16 and c18 fatty acids, resulted in a marked increase in exosome secretion. in addition, lysosomal cer accumulation induced by acid ceramidase inhibition also accelerated exosome production. knockdown of laptm4b significantly prevented the ceramide-dependent exosome release. in addition, we showed that these cer loading promoted colocalization of cd63-positive mvb with rab11-positive recycling endosomes, further demonstrated that laptm4b knockdown cancelled the cer-dependent increase of the colocalization. summary/conclusion: these data suggest that lysosomal cer binds to laptm4b and promote the transport of mvb to plasma membrane, resulting in an increase of exosome secretion in neuronal cells. chloroquine-mediated lysosomal inhibition alters composition and function of cancer-derived extracellular vesicles jing xu a , kevin yang a , shane colborne a , elham hosseini-beheshti b , gregg morin a , emma guns b and sharon m. gorski a a bc cancer, vancouver, canada; b the vancouver prostate centre, vancouver, canada introduction: small extracellular vesicles (sev) are signalling entities released by many types of eukaryotic cells. sev are of special interest in cancer due to their reported roles in modulating the cancer microenvironment and facilitating cancer cell invasion. macroautophagy (hereafter autophagy) is a catabolic process well-known for the recycling of cytosolic cargos through lysosome-mediated degradation. in this study, we profiled the changes in sev content and function under lysosome inhibition and investigated the involvement of autophagy machinery in sev content. methods: chloroquine (cq) was used to inhibit lysosomal degradation and autophagy turnover in triplenegative breast cancer (tnbc) cell lines. sev were collected via precipitation after pre-clearing and concentration of conditioned media. western blotting, nanosight and transmission electron microscopy were used to profile sev. quantitative mass spectrometry was used to characterize cq-induced changes in the sev proteome. antibody-conjugated magnetic beads were used in immunoprecipitation of sev. results: cq treatment did not substantially alter the physical properties of tnbc-derived sev. however, cq treatment altered the sev proteome and growth effects of sev on normal and endothelial recipient cells. cq treatment induced co-localization of mammalian atg8 proteins with endolysosomal markers in the cytoplasm, which coincided with an enrichment of atg8 s and their adaptor proteins in sev. cq-induced enrichment of atg8 s in sev required lipidation, and occured preferentially in one subset of sev. summary/conclusion: our study reveals changes in the content and function of cancer cell-derived sev in response to perturbation of intracellular trafficking pathways, demonstrates the flexibility and heterogeneity of sev composition, and has implications for cq efficacy in therapeutic settings. introduction: introduction: argonaute 2 (ago2) is the essential component of the rna-induced silencing complex (risc) that binds mirnas and promotes mrna degradation. extracellular vesicle (ev)-carried mirnas have been shown to influence gene expression and functional phenotypes in recipient cells. many investigators have found ago2 in evs and it is postulated that ago2 is a major transporter of mirnas into small evs (sevs), such as exosomes. others have reported extracellular ago2 that is non-vesicular. we set out to evaluate the effect of growth factor signalling and serum contamination on the detection of ago2 in sevs. methods: methods: wildtype kras colorectal cancer cells, dks8, were conditioned with 3 different culture media (serum-free dmem, dmem supplemented with ev-depleted fbs, and opti-mem). evs were purified from conditioned media by cushion-density gradient ultracentrifugation. western blot analysis of dks8 total cell lysates, large evs and density gradient fractions was performed, probing for ago2 and ev marker proteins. the size and concentration of the evs were determined by particle metrix analysis. results: results: in all conditions, we found the highest abundance of sevs in fractions 6 and 7, as assessed by western blot analysis. ago2 was detected in the same fractions as sevs in both the serum-free dmem and opti-mem conditions, although the levels of ago2 was higher in the serum-free dmem fractions compared to that of opti-mem. in contrast, ago2 was present in both vesicular and non-vesicular fractions in the dmem supplemented with ev-depleted fbs condition. no significant differences were observed in the size and number of evs collected in the three conditioning methods. summary/conclusion: summary/conclusion: the presence or absence of ago2 in evs has been controversial. multiple factors may affect the ability to detect vesicular ago2, including serum and growth factors in the conditioned media that may provide sources of extravesicular ago2 and also regulate the trafficking of ago2 into vesicles. introduction: cancer-associated glycosphingolipids have been utilized as tumour markers and targets of cancer therapy. we have investigated roles of gangliosides in cancers, and clarified that cancerassociated gangliosides enhance malignant properties of cells by forming complexes with membrane molecules in lipid rafts. in this study, we analysed contents of gangliosides and membrane molecules on extracellular vesicles (ecvs) secreted from melanoma cell lines. methods: melanoma cell lines with various ganglioside patterns were used for isolation of ecvs. gangliosidemodified melanomas with genetic engineering were also used. genetic modification was done by cdnas of ganglioside synthase genes. ecvs were collected by ultra-centrifugation, or by tim4-beads. contents in ecvs were analysed by immunoblotting or flow cytometry. roles of lipid rafts in the generation and secretion of ecvs were analysed by treating cells with 1 mm methyl β-cyclodextrin. results: using 4 melanoma cell lines, ecvs were isolated by ultra-centrifugation, and their sizes were analysed by nanosight. all samples showed uniform sizes between 100 and 200 nm. protein amounts in ecvs were measured, showing heterogeneous levels at 10~80 μg/100 ml. then, gangliosides expressed on ecvs from these cell lines were analysed using tim4beads and flow cytometry. gd3 and gd2 were detected on ecvs almost proportionally with expression levels of those gangliosides on the cell surface. then, immunoblotting was performed to analyse integrin levels in ecvs from transfectant cells expressing high levels of gd3, showing increased levels of integrins in ecvs from gd3+ cells compared with those from gd3-cell lines. integrin levels in cell lysates from these cells (gd3+ and gd3cells) were almost equivalent. treatment of a gd3-expressing melanoma cell line by 1 mm methyl β-cyclodextrin resulted in marked reduction of secreted ecvs and amounts of tsg101 in them. summary/conclusion: ganglioside expression patterns on melanoma cells were well reflected in the expression of gangliosides on ecvs. these results as well as increased levels of integrins in ecvs from gd3 + cells suggest that gangliosides and lipid rafts are involved in the generation and secretion of ecvs. introduction: hypoxia, or low oxygen tension, is a common feature associated with tumour growth and is known to regulate tumour cell function, especially through rewiring of cell metabolism. however, how hypoxia influences tumour cell interactions with surrounding cells is not fully elucidated. we sought to evaluate how hypoxia alters metabolite and metabolism-associated mirna packaging in exosomes. methods: exosomes were isolated from 4t1 breast cancer cells cultured in normoxia (21% o2) and hypoxia (5% o2) via ultracentrifugation, optiprep gradients, and size exclusion chromatography. exosomes were further characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. metabolite and mirna profiling was performed on exosomes and exosome-producing cells in normoxia and hypoxia. results: secretion of exosomes was increased under hypoxic conditions. metabolite profiling revealed alterations in metabolites specific to exosomes derived from hypoxic cells. profiling of exosomal mirna showed packaging of metabolism-related mirna into exosomes derived from hypoxic cells. summary/conclusion: hypoxia alters the metabolite and mirna profiles of cancer cells, with selective packaging of these molecules into exosomes. we identified metabolites and mirna that are depleted and enriched in exosomes compared to cells. these studies identify hypoxia-associated shifts in exosome cargo, providing insight into exosome cargo packaging with potential implications for understanding how cancer cell-derived exosomes regulate recipient cell function. lysosomotropic agents prompts the release of extracellular vesicles carrying autophagy-associated markers: evidence of a general mechanism of secretion driven by lysosomal impairment introduction: drug-induced lysosomal storage disorders (lsds) are due to the transient intracellular accumulation, mostly of phospholipids, into multilamellar inclusion bodies within late endosomal/lysosomal compartment. they represent a major side-effect for many drugs of several pharmacological categories. most lsds inducers are cationic amphiphilic drug (cad), but the molecular mechanisms leading to accumulation of undigested substrates are unknown. extracellular vesicles (evs) have been implicated in cell waste disposal, but it is unclear whether they might be involved in extracellular release of undigested substrates. methods: to investigate this aspect, we developed hek cells stably expressing the fluorescent fusion proteins egfp-cd63 and mcherry-cd63, separated evs by differential ultracentrifugation and quantified by evassociated fluorescence and nta particle count. results: evs released by these models upon treatment with drugs inducing the accumulation of phospholipids (amiodarone) or glycosaminoglycans (tilorone), showed the release of fluorescent medium/large evs (10k fraction) and small evs (100k fraction), whose size and distribution were similar to the same vesicles released by control cells, but enhanced the recovery of medium/large evs and to a lower extent of small evs, analysis of evs associated markers revealed a dosedependent increase of autophagy-associated markers in medium/large and small evs. similar results were obtained when autophagic flux was impaired by drugs raising lysosomal ph by different mechanisms, such as chloroquine and bafilomycin, but not when autophagic flux was stimulated by drugs such as curcumin or overexpression of the endosomal/lysosomal regulator tfeb. summary/conclusion: overall results show that impairment of autophagic flux, either by indigested substrates or higher lysosomal ph, is associated with an increased release evs enriched in autophagy markers, compatible with autophagomes and/or amphisomes, unravelling a connection with secretory autophagy. tomofumi yamamoto a , yusuke yamawaki b , yutaka hattori c and takahiro ochiya a a tokyo medical university, shinjuku-ku, japan; b national cancer center research institute, chuo-ku, japan; c keio university faculty of pharmacy, minato-ku, japan introduction: multiple myeloma (mm) is a haematological tumour. last decade, the prognosis of mm has improved by the development of therapeutic drugs; however, mm cells acquire drug resistance by longterm exposure of these therapeutic drugs. one of the possible explanations of drug resistance is that cells with drug resistance transmit information to other mm cells and their microenvironmental cells. although the elucidation of the mechanism of drug resistance in mm have been desired, it remains poorly understood. methods: in order to understand the mechanism of drug resistance in mm, lenalidomide resistant cell lines were established by long-term exposure of low concentration of lenalidomide. drug resistance was assessed by mts assay and caspase assay. the amount of ev was measured by exoscreen, which is ultra-sensitive detection method of evs by measuring surface protein of evs, such as, cd9 and cd63 (yoshioka et al., nat commun., 2015) . to identify the genes which involved in drug resistance, rna sequence among the drug-resistant cell lines and their parental cell lines was performed. results: firstly, characterization of these cells was confirmed. we found that all of the lenalidomide resistant cell lines secreted more evs than their parental cell lines. in addition to this, the size of ev derived from resistant cells are smaller than those of parental cells. next, we collected evs from resistant cells and parental cells by using ultracentrifugation, and added them to parental cells in the presence of lethal dose of lenalidomide. compared with ev derived from parental cell lines, the evs derived from lenalidomide resistant cell lines increased a number of living parental cells. these results suggested that the evs derived from lenalidomide resistant cells can affect the lenalidomide sensitive cells. as a result of rna sequence, several genes highly expressed in resistant cell line we found, which associated with lysosome pathway. among them, attenuating the sort1 and lamp2 genes could significantly reduce the ev secretion in mm cells, leading to enhance the lenalidomide sensitivity. summary/conclusion: our results showed that ev secretion via sort1 or lamp2 could induce the drug resistance in mm. study on biological stimulate mechanism of stem cell-derived exosome generation by nanoparticles introduction: mesenchymal stem cells (mscs) are pluripotent stromal cells known to release extracellular vesicles (evs) containing various growth factors and antioxidants that can positively affect surrounding cells. nanoscale msc-derived evs, such as exosomes, have been developed as bio-stable nano-type materials, but had low yield and were difficult to quantify. we hypothesized that the mechanism of nanoparticleenhanced exosome production would stimulate intracellular molecules. the aim of this study was to elucidate the molecular mechanisms of exosome generation by comparing the internalization of surface-modified positively charged nanoparticles and exosome generation from mscs. methods: mesenchymal stem cells (mscs) were cultured in mem-alpha with 10% fbs and 1× antibiotics. the positively charged nanoparticles were synthesized by poly-lactide-co-glicolide (plga) and polyethylenimine (pei) with cy5.5 for tracking nanoparticles. all of the exosome image were identified using an electron microscope. additionally, it was confirmed the internalization of the nanoparticles by if. the primary antibodies used were anti-eea1, anti-rab7 and anti-gm130. in order to prove the development of exosomes, rt-pcr using autophagy-related mrna was performed. real-time rt-pcr was performed using the applied biosystems sequence detection system 7900. lastly, mirna from msc-derived exosome analysed automatically in the affymetrix data extraction protocol using the provided affymetrix genechip® command console® software (agcc). all statistical testing and visualization of differentially expressed genes was conducted using r statistical language 3.3.3 results: we determined that rab7, located in the mvb and autolysosomal membrane, was increased upon exosome expression and was associated with autophagosome formation. these results suggested that nanoparticles migrated to lysosomes during treatment; however, intracellular exosome-forming factors were stimulated during endosomal maturation simultaneously. summary/conclusion: therefore, msc-derived exosome research using nanoparticles is useful for increasing exosome yield and the discovery of nanoparticleinduced genetic factors. theoretical description of formation of extracellular vesicles by budding of membrane introduction: understanding mechanisms of extracellular vesicles (evs) formation is of utmost importance for their effective use in science, medicine and technology. in particular, the discovery of universal mechanisms explaining the phenomena taking place in vesiculation appears to be crucial and highly warranted. mammalian erythrocytes and giant phospholipid vesicles have been largely used as model systems to study principles of membrane budding and vesiculation. the mechanisms conveniently studied in these simple systems are then generalized to other types of biological membranes. we present a theoretical description of membrane budding and compare the theoretically obtained shapes with the observed ones. methods: in accordance with the fluid crystal mosaic model, membrane is considered as composed of constituents (inclusions) subjected to the local curvature field created by surrounding constituents. constituents can attain different in-plane orientations in the membrane which correspond to different energies. the thermal motion oposes the complete orientational ordering. the single-constituent energy expresses a mismatch of the curvature of the membrane at the position of the constituent and the intrinsic principal curvatures of the constituent and inplane orientation of their principal axes. the free energy of the whole membrane is obtained by summing up (integration) the contributions of the constituents and using methods of statistical physics, and minimized by using numerical methods. results: to outline the principle of (outward and inward) budding, respective sequences of shapes corresponding to a formation of one (outward and inward) spherical bud were calculated by minimization of the free energy. also the corresponding shapes observed in evs (imaged by electron microscopy) and in erythrocytes and giant phospholipid vesicles (imaged by optical microscopy) are shown. it can be seen that theoretically calculated shapes and experimentally observed ones agree well over up to 3 orders of magnitude (the order of the size of giant phospholipid vesicles is between 1 and 100 micro metres, in erythrocytes it is about 5 micro metres and in evs it is about 100 nanometres). summary/conclusion: budding of the membrane is an universal mechanism in formation of external and internal vesicles. introduction: the release of extracellular vesicles (evs) from cells is important for many cellular mechanisms both in normal physiology and in disease. arrdc4 (arrestin domain containing protein 4) is an adaptor protein known to facilitate the ubiquitination of target substrates by nedd4 family ubiquitin ligases. it also traffics cargo to extracellular vesicles. previous studies show the involvement of arrdc4 in the trafficking of the divalent metal ion transporter dmt1 to evs in a ubiquitin-dependent manner, and we aimed to further understand this mechanism. methods: we performed mass spectrometry to identify ubiquitinated lysine residues in arrdc4. we then generated arrdc4 wt and lysine mutant clones and expressed these in cells to determine the effect on ev biogenesis and protein trafficking. results: mass spectrometry data identified 5 potential ubiquitinated lysine residues. out of these, lysine 270 appeared to be the most important for arrdc4 function. arrdc4k270 r mutation caused a decrease in the number of ev released by the cell compared to arrdc4 wt, and a reduction in trafficking of dmt1 to evs. furthermore, we also observed a decrease in dmt1 activity and an increase in its intracellular degradation in the presence of arrdc4k270 r. k270 also appeared to be ubiquitinated with k29 polyubiquitin chains by the ubiquitin ligase smurf1. summary/conclusion: our data suggests that k29 polyubiquitin chains are the signal for arrdc4mediated ev biogenesis and protein trafficking, and loss of this signal causes cargo to be rerouted to intracellular degradation mechanisms. chair: tanina arab -department of molecular and comparative pathobiology, johns hopkins university school of medicine a 3d-printed model to represent the structure and nature of extracellular vesicles, for public engagement and education events. christian burton a , sara veiga a , jason webber a , kate milward a , muireann ni bhaoighill a , lauren evans a , andreia de almeida b , rachel j. errington a and aled clayton a a cardiff university, cardiff, uk; b cardiff university, research associate, uk introduction: explaining the field of extracellular vesicles to the lay public and young audiences can often be challenging. whilst diagrams and images of evs may be helpful, conveying clearly the shape and composition of an ev by these means is not always a success. whilst many members of the audience may be familiar with concepts of cells and related structures, others will find such discussions very abstract and challenging. in order to aid interactions with lay audiences we embarked on the design of a physical hand-held plastic model, representing a typical ev. incorporating flexibility in the design allowing the community to adapt it to showcase their own research. the second goal was to ensure manufacturability using widely available 3dprinting technologies. methods: the basic model design was conceived by dr c. burton, and iteratively developed using solidworks, 2019, then exported for use in any cad environment (stl format). a model showing a halved ev hemisphere, with a visible lipid-bilayer was developed. attachable rings allow trans-membrane-molecules to be represented, current designs include mhc class-i, hspgs, integrins, tetraspanins and supported by handouts accompanying the models. intraluminal cargo is included via removeable "pegs", and examples representing rna or simple globular proteins, and a template has been created. results: the design is free and open source, and available to the community at: https://www.thingiverse. com/thing:3986565. instructions for 3d printing are available from the uk extracellular vesicle society website; https://www.ukev.org.uk/public-engagementmaterials/. models have been produced using entrylevel 3d printers and trialled at engagement events with good early responses. summary/conclusion: the authors hope the community will use and develop this 3d-model design and that the approach provides an additional and helpful tool for educating audiences about the complexities and roles of evs in biology and disease. centrifugal filtration-sec is promising for extracellular vesicle isolation from d492 and d492her2+ breast epithelial cell lines introduction: despite recent developments in breast cancer therapy, there is still need for a more targeted approach. extracellular vesicles (evs), endogenous nanovesicles released from human cells, are an attractive choice as nanodrug carriers due to their size, stability and their unique targeting specificity. the aim of this study was to determine if centrifugal filtration (cf) combined with size exclusion chromatography (cf-sec) would be useful for ev isolation from two epithelial breast cell lines d492 and d492her2+, representing the tissue of interest, and the amount of cell culture needed to get measurable ev concentrations. methods: cell culture media (without serum) from the immortalized breast epithelial cell lines d492 and d492her2+ was concentrated with centrifugal filtration (cf) followed by isolation with size-exclusion chromatography (sec) using hiprep 16/60 sephacryl s-400 column run with äkta start (280 nm), 240 min runs. each fraction (4-5 ml) was collected with fraction collector. dulbecco's particle free pbs was used as mobile phase. the resulting particles were analysed with nanoparticle tracking analysis (nta, nanosight ns300, camera gain 10, static mode, capture time 90 sec), western blotting (wb), microbca and transmission electron microscopy (tem, samples fixed with 2% formaldehyse and stained with 2% uranyl acetate, run at 80kv). results: although sec did not show any prevalent peaks from early eluting regions previously shown to contain extracellular vesicles, these fractions (f1-f3, 40-130 min) were collected from d492her2+ cell culture medium. interestingly, both nta and tem suggest that f2 and f3 contained evs as the isolated particles measured 65 and 58 nm, respectively and tem revealed spherical particles 20-50 nm in diameter. wb was unable to detect the ev associated protein alix (but was present in the whole cell lysate). soluble proteins and protein aggregates eluted late in the sec chromatogram (180 min), with protein analysis (microbca), tem and wb confirming their presence. summary/conclusion: cf-sec is a promising method for ev isolation for pharmaceutical applications, but further work is needed to optimize the isolation process using äkta start for these cell lines. customer stories from the ev core of university of helsinki introduction: the ev core, world's first ev-dedicated technology platform established in 2016, is a joint venture of two extracellular vesicle (ev) research laboratories at university of helsinki. as an academic research/service facility, the ev core provides infrastructure, state-of-the-art and emerging ev-technologies for research groups, hospitals, companies and authorities in the ev-field. the ev core provides ev isolation, purification and characterization services and offers contacts to downstream analyses in other core facilities based on optimized ev protocols. here, we present and discuss the customer experiences and prospects with the aim to further develop ev core services. methods: our most wanted services are nanoparticle tracking analysis, electron microscopy, ev isolation and rna isolation and consultation. currently, the key down-stream analysis methods are (mi)rna sequencing, metabolomics, flow cytometry and functional assays. results: we present the stories from our customers starting with their research questions and need for the ev expertise/consultation and equipment. next, we show how the projects advanced and what types of ev core -derived or other downstream services helped them to achieve their aims. in the end, we will acknowledge the customers experience and current status of their research. summary/conclusion: narratives of customer stories are an effective starting point for fruitful discussions about the current status and next developments in the young ev service field. recent isev workshops: open, reproducible and standardized ev research (ghent, 2019) and evs in immunology (buenos aires, 2020) introduction: since its founding in 2012, isev has sought to further extracellular vesicle research in various ways including scientific meetings. these events encompass annual meetings as well as smaller, topically focused workshops, with the first isev workshop (on rna and evs) organized in new york city in october, 2012. in december, 2019, the workshop "open, reproducible, and standardized ev research" was held in ghent, belgium. in march, 2020, the workshop, "evs in immunology" was held in buenos aires, argentina, with a preceding education day. methods: the international organizing committees of the 2019 and 2020 isev workshops prepared scientific programs around key themes of ev rigour and standardization (ghent, belgium, 2019 workshop) and evs in immunology (buenos aires, argentina, 2020 workshop). abstract and application submissions were invited. applications were reviewed and ranked by panels of ev experts for each event, and participants were invited. results: the 2019 and 2020 workshops assembled a total of more than 100 individuals for talks and discussions around the themes of rigour and standardization and evs in immunology. the buenos aires workshop was preceded by an education day, coordinated by the isev executive committees for education and science and meetings. during these two workshops, poster presentations were permitted for the first time, affording additional presentation and interaction opportunities. the rigour and standardization workshop also featured real-time discussant polling to facilitate discussion. summary/conclusion: isev workshops such as those addressing rigour and standardization (ghent, 2019) and evs in immunology (buenos aires, 2020) continue to provide opportunities for focused discussion of small groups of experts on key topics in the field. often followed by published products, isev workshops help to lead and coordinate progress in ev science. for future isev workshops, educational activities may again expand the reach of each event, while poster sessions and app-driven real-time responses should be considered for enhanced interactions and participant canvassing. ev journal club: exchanging pizza for a worldwide audience during covid-19 kenneth w. witwer johns hopkins university school of medicine, baltimore, usa introduction: a monthly journal club focused on extracellular vesicle science was established at johns hopkins university in 2016, featuring lunch and presentations by academic and industry participants. when covid-19 prevented in-person meetings beginning in march, 2020, the journal club was converted to a virtual, weekly format on the popular online meeting app zoom. the journal club has persisted despite initial problems with online vandalism. most sessions are also made public on a youtube channel, https:// www.youtube.com/c/extracellularvesicleclub. methods: weekly ev club sessions are arranged by the host. most focus on a specific manuscript related to evs, but some weeks feature presentations of published or soon-to-be-published research by the presenting authors. sessions are advertised one week to several days in advance on social media platforms such as linkedin, twitter, and facebook, asking interested parties to sign up to join a mailing list via surveymonkey. the log-in information is then sent to the mailing list. upon clicking the link, participants are placed in a virtual waiting room for vetting by the host and volunteers. after admission, all parties but the host and presenter are muted to avoid distractions. questions and comments may be placed in a chat box. contributions are monitored and compiled by the host and volunteers to build a question-and-answer session at the end of the presentation. recorded sessions-with or without editing as needed-are placed on the youtube channel for additional access. results: despite initial problems with online vandalism known as "zoombombing," the journal club has continued weekly during the covid-19 shutdown in the host country (us). an audience of between 80 and 150 individuals is typical. participants typically ask more questions than can be answered in a one-hour time frame. the online format also allows for debate-style events and polling of the audience. summary/conclusion: this ev journal club is an example of how online tools can be used to facilitate international scientific interactions. further development of such formats could provide alternative approaches for isev activities in the science, education, and communication areas. the study aim is to assess whether the exposure to pm10 and pm2,5, chosen as paradigmatic environmental stressors, could modify the composition of nasal microbiota (nm) and extracellular vesicle (ev9 signalling network, showing a role in allergic ar exacerbation). methods: nm analysis were performed on v3-v4 16 s rrna gene regions amplified from upper-airway tracts of 25 ar cases and 25 healthy individual controls to perform nm analyses. ev size, concentration and cellular origin for each subject were assessed by nanoparticle tracking analysis (nta) and flow-cytometry (fc). information on daily pm10 and pm2,5 concentrations at the municipality of residence in the 7 days preceding nasal sampling (i.e. day −1 to day −7) was assigned to each subject by arcgis software. multivariable and logistic analyses were applied on nm, nta and fc outcomes. results: when taxonomy composition was considered, in controls actinobacteria (50.8%) was the most represented, followed by firmicutes (34.7%) and proteobacteria (12.8%) while in cases proteobacteria were 38.8%, actinobacteria were 37.1% and firmicutes were 23.4%. cases showed a higher concentration of all the investigated ev types, derived from platelets (cd61 +), activated endothelium (cd62e+), monocytes (cd14+), eosinophils (cd294+), neutrophils (cd177+), mastocytes (cd203 c+), epithelial cells (epcam+), gram+ bacteria (lipoteichoic acid+), gram-bacteria (lps+). the effect was greatest in the case of mastocytes evs which were increased 2.5fold in cases versus controls (p < 0.001). evs were modified by pm exposure at several time lags. in particular, a negative association between pm10 and eosinophil evs was observed (beta = −0,016; pvalue = 0,017). as we clustered subjects according to their nm, we observed this variable was a strong effect modifier of the association between pm exposure and ev release. summary/conclusion: our findings start to provide an insight on the effect of air pollution on evs, taking into account the effect of nm, in patients with ar. further research is necessary to disentangle the mechanism exerted by inhaled pollutants in modulating evs and nm, and therefore ar exacerbation. funding: gsk investigator sponsered study aryl hydrocarbon receptor activation induces the expression of specific microrrnas in th17 cells that are release into extracellular vesicules and associated with arthritis introduction: in rheumatoid arthritis (ra), an autoimmune disorder characterized by a chronic sinovial inflammation, smoking is a major risk factor contributing to disease progression, and poor response to therapy. th17 cell is actively involved in worsening smooking-associates inflammation mediated by aryl hydrocarbon receptor (ahr), a cytoplasmic transcription factor involved in xenobiotic metabolism. both, ahr and th17 cells, has important implications during ra development. considering that cigarette smoke is a potent epigenetic modifier, we hypothesized that ahr activation, by cigarette components, would transcribe specific micrornas in th17 cells as a molecular mechanism to exacerbate inflammation in arthritis. methods: microrna expression was evaluated by largescale approach or real-time pcr. c57/bl6 and ahr null mice were submitted to arthritis experimental models and exposed or not to cigarrete smoke (ethical committee approved 048/2012). extracellular vesicles (evs) were isolated by ultracentrifugation, and characterized by western blot and nanosight. rankl-induced osteoclasts (ocs) differentiation in vitro was stained for trap. inhibition of mirnas were performed using anti-mirs transfection. results: we identified a specific group of mirnas induced in th17 cells after ahr activation. during arthritis progression, the micrornas are expressed and increases after exposure to cigarette smoke. in the absence of ahr their levels were drastically reduced. interestingly, we found that these micrornas are released by th17 cells into evs, and are able to promote osteoclastogenesis. ocs differentiation in vitro increases in the presence of th17-derived evs, and this process is reduced in the absence of micrornas. summary/conclusion: microrna-mediated gene regulation plays crucial roles in the immune system functions, and their abnormal expression is highly correlated with the pathogenesis of ra. evs are known to function in cell-to-cell communication and are able to transmit their contents and cause changes in the target cell. our findings demonstrate a new molecular mechanism by which cigarette smoke could aggravate inflammation in arthritis; through the activation of ahr receptor in th17 cells, inducing the transcription of specific micrornas that are released into evs, and act as pro-inflammatory mediators. introduction: chagas disease (cd) is caused by the flagellated protozoan t. cruzi. trypomastigote forms are capable of releasing extracellular vesicles (evs) that contain the major surface molecules of the parasite. the parasite has a complex life cycle that leads to it a rapid adaptation in the environmental changes in the hosts. however, the effects of stress on on evs release are not completely understood. objetive: we evaluated the release of evs by trypomastigotes incubated under different stress conditions and the immunomodulatory role of these evs in pre-activated bone marrow-derived macrophages (bmdm). methods: nanoparticle tracking analysis (nta) and scanning electron microscopy (sem) showed an increase in evs releasing by trypomastigotes at 4°c under acidic conditions, evs released was affected and triggered amastigogenesis process. results: treatment with sodium azide (nan3) also caused changes in the release of evs regarding size and concentration. nitrosative stress caused by sodium nitrite (in culture medium mildly acidic, ph 5.5; in this condition nano2 releases nitric oxide) stimulated an increase in production of evs by t. cruzi. when the parasites were treated with 100 nm s-nitrosoglutathione (snog), we observed a reduction in size and concentration of vesiculate material by trypomastigotes. at a higher snog concentration (100 µm), the concentration of the vesiculate material increased. t. cruzi-derived evs exposed to stress conditions increased the expression of inos, arg 1, il-12 and il-23 genes in ifn-γ and lps pre-activated bmms. summary/conclusion: results suggest that the viability and/or integrity of the parasite are necessary for the evs releasing. in those in vitro conditions they triggered a proinflammatory response in host cells. this may be a strategy developed by the parasite to favour its establishment in the host. funding: fapesp, cnpq, capes and fapemig ppm-x 00102/6. immuno-toxicological evaluation of human mesenchymal stem cellintroduction: mesenchymal stem cells (mscs) have been widely used to the field of autoimmune diseases or tissue regeneration therapy. recently, many research groups have reported that mscs showed their ability via secreted paracrine mediators including extracellular vesicles (evs) rather than cell-to-cell contact. mscs mainly exist on bone marrow, peripheral blood, umbilical cord and adipose and can mostly secrete evs. it has emerged that evs alone are responsible for the therapeutic effect of mscs in plenty of animal diseases models. hence, msc-derived evs may be used as an alternative msc-based therapy in regenerative medicine. methods: as part of safety programme for human therapeutics, we performed immunotoxicological assessment of evs obtained from human mscs (hevs) in mice and human peripheral blood mononuclear cells (hpbmcs). firstly, mice were treated intravenously with a negative control, a positive control (lps; 0.4 mg/kg), or low-dose (1x10e8 paticles/head) and high-dose (1x10e9 paticles/ head) of hevs every other day for 10 days and then analysed lymphocyte subsets from collected spleen by facs. next, we treated the evs on hpbmcs for 3 days with low conc. (1x10e8 particles/ml), high conc. (1x10e9 particles/ml), pma/ionomycin as a cell activator or cpt (10 μm) as an apoptotic inducer. annexin v/pi and csfe were analysed by facs. results: as a result, splenic nk cells and b cells were slightly increased about 2~7% in hevs-treated mice, without biological significance, compared with a positive control (lps) as an immunogenicity inducer. and there were no effects on serum levels of inflammatory cytokines in mice. in addition, hevs had no cytotoxic effect on hpbmcs at both low and high conc. under the culture medium with evs-depleted fbs, the hevs appeared minimal anti-apoptotic effect on hpbmcs. for the cfse assay, the hevs showed slight proliferation on hpbmcs and pbmc activation induced by pma/ionomycin. summary/conclusion: in conclusion, the hevs have little immuno-toxicological effects in mice and hpbmcs. further detailed studies to elucidate immunological response of hevs for development of human therapeutics are needed. funding: this research was supported by a grant (18172mfds173) from ministry of food and drug safety. investigation of immune response to mesenchymal stem cell-derived extracellular vesicles in the cancer setting introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) are thought to be a fingerprint of the secreting cell and therefore may retain the cancer targeting and immune privilege of mscs. thus msc-evs hold immense potential as tumour-targeted therapeutics for breast cancer. the aim of this study was to determine whether msc-ev administration in tumour bearing immunocompetent animals would initiate an immune response. methods: evs were isolated from conditioned media of both human and murine bone marrow-derived mscs through sequential differential centrifugation, microfiltration and ultracentrifugation. evs were characterized by nanoparticle tracking analysis (nta), western blot and transmission electron microscopy (tem). 1 x 10(8) human or murine msc-evs were administered intravenously into 4t1 breast tumour bearing balb/c mice (n = 6) and healthy controls (n = 6). tumour tissue, draining lymph node and spleen were then harvested, dissociated and flow cytometry performed targeting markers associated with a range of immune cells including t-cells, macrophages and natural killer (nk) cells. results: evs were successfully isolated from murine and human mscs with the appropriate size of small evs (sevs: 30-150 nm) and morphology including a lipid bilayer observed by tem. evs expressed tetraspanins cd63, cd81, cd82; cytosolic protein tsg101 and were negative for calnexin. ev concentrations ranged from 4.08 x 10(9) -6.6 x 10(9)/ml. in order to study a range of immune cell populations two antibody panels were created using complimentary fluorescent dyes. the proportion of t-cells (cd4+, cd8+, cd25+), neutrophils (gr-1+, ly-6 c+), dendritic cells (cd11 c+), macrophages (cd11b+, mhci+, mhcii+), nk cells (cd27+) and b cells (cd19+) remained stable in the tumour, draining lymph node and spleen of all tumour-bearing animals that received either human or murine msc-evs, with no significant change observed in any category. summary/conclusion: the data presented supports the hypothesis that msc-evs retain the immune privilege of the secretory cell, with human cell-derived evs illiciting no immune response in mice. this is encouraging and reinforces the potential for use of msc-evs in the therapeutic setting. introduction: mycobacterium avium (m. avium) is a slow growth rate non-tuberculous mycobacterium (ntm). m. avium infection is a severe global health problem. but the mechanisms of pathogenicity of m. avium are poorly understood. outer membrane vesicles (omvs) that traverse the cell wall and contain a varied bioactive components inculding dna, rna, protein and toxins. previous studies have suggested that these omvs are produced in vitro and during animal infection, but the role of omvs secretion during the interaction of m. avium with host cells remains unknown. methods: in this study, m. avium were grown in middlebrook 7h9 medium (m7h9) supplemented with 10% (v/v) oadc enrichment and 0.5% (v/v) glycero. m. avium omvs were isolated by ultracentrifugation method. characterization of omvs by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). the raw 264.7 murine macrophages were incubated with the m. avium omvs to analyse inflammatory response and production of nitric oxide (no) and reactive oxygen species (ros) of macrophage. results: in this study, we demonstrate by fluorescence microscopy that murine macrophages can phagocytosis omvs produced by m. avium. incubation of m. avium omvs with murine macrophages resulted in increased levels of extracellular tumour necrosis factor alpha (tnf-α), interleukin-1β (il-1β), terleukin-6 (il-6) and interleukin-12 (il-12). meanwhile omvs stimulated macrophages produce no and ros. introduction: hospital associated venous thromboembolism (ha-vte) in paediatric patients is the second most common contributor to harm in hospitalized children. platelet-endothelial interactions are integral to the formation of vte, especially in inflammatory conditions such as sepsis. small extracellular vesicles (sevs) have the ability to reprogramme target cell phenotypes via their microrna contents and are known to contribute to vte formation. we hypothesize that sepsis alters platelet-derived sev micrornas capable of net upregulation of vascular endothelial procoagulant and downregulation of anticoagulant pathways. methods: using a precipitation solution and size exclusion chromatography, we isolated sevs from platelet poor plasma of children admitted to the paediatric intensive care unit for sepsis and from healthy controls. we positively selected platelet-derived sevs using immunomagnetic isolation for cd42b platelet antigen and confirmed selection using flow cytometry. microrna was profiled using affymetrix genechip mirna 4.0 array. results: microrna from 9 sepsis patients (median age 7.0 years; iqr:1.2-13 and 77% female) with a median psofa score of 6 (iqr:2.5-10) and from 5 healthy controls (median age 9 years; iqr:6.5-13.5 and 20% female) was isolated and compared. in septic vs. healthy patients 30 mirnas were differentially expressed (false discovery rate (fdr)<0.05; fold change ≥|1.5|) affecting 921 mrna pathways. in septic children, pathways affecting chemotaxis and cell movement of leukocytes were predicted to be activated with z-scores ≥2. summary/conclusion: we developed a method to successfully isolate platelet-derived sevs. sepsis alters the platelet-derived sev microrna profile in paediatric patients with sepsis. these micrornas are predicted to target chemotaxis and cell movement pathways, important contributors in the formation of ha-vte. further analysis into specifically targeted pathways should be conducted as a potential target for the prevention of ha-vte in sepsis. introduction: sjögren´s syndrome (ss) is a systemic autoimmune disease that mainly affects salivary and lacrimal glands. mechanisms of ss pathogenesis are poorly understood. it is thought that inflammation leads to destruction of exocrine glands, however the triggers of autoimmunity and the mechanisms by which inflammation drives immunopathology are not characterized. our work identifies t cell-exosomederived mir-142-3p as a pathogenic driver of immunopathology in ss. micrornas (mirnas) are endogenous small noncoding rna molecules that regulate the expression of target genes through translational repression of mrnas. through transcriptomic profiling studies our group had previously documented a significant upregulation of mir-142-3p in patient ss tissues and in serum exosomes. methods: structured search for target genes of mir-142-3p involved in salivary gland (sg) physiology was performed with mirdip 4. serca2b, ryr2 and ac9 were selected for further validation and functional analysis. binding of the mirna was confirmed by luciferase reporter assays in hsg cell lines and human-derived primary epithelial cells. the mrna and protein levels of serca2b, ryr2 and ac9 were determined by qpcr and western blot, respectively. to investigate the cell-specific distribution of mir-142-3p in relation to the expression levels of serca2b, ryr2, and ac9, a double fluorescent in situ hybridization was performed. ca2+ signalling and camp levels were measured using fluorescent sensor. to isolate exosomes, the t cell medium and serum of ss-patients and healthy volunteers (hv) were collected. results: we show that mir-142-3p is over-expression in the sgs of ss-patients. next, we demonstrated that mir-142-3p is contained in exosomes in serum of sspatients significantly more than serum of hv. we also show that activated t cells secrete exosomes containing mir-142-3p which transfer into glandular cells and affecting intracellular ca2+ signalling, camp production and protein production by mir-142-3p targets (serca2b, ryr2 and ac9). summary/conclusion: this study provides evidence for a functional role of the mir-142-3p in ss pathogenesis and promotes the concept that t cell-activation directly may impair epithelial cell function through secretion of mi-rna containing exosomes. treg-derived il35-coated extracellular vesicles promote infectious tolerance (p35) subunits, yet the forms that il35 assumes and its role in peripheral tolerance, remain elusive. methods: we induce cba-specific, il35-producing t regulatory (treg) cells in tregebi3 wt c57bl/6 reporter mice, and identify il35 producers by expression of ebi3tdtom gene reporter, plus ebi3 and p35 proteins. results: curiously, both subunits of il35 were displayed on the surface of tolerogen-specific foxp3 + and foxp3neg (itr35) t cells. furthermore, il35 producers, although rare, secrete ebi3 and p35 on extracellular vesicles (ev) targeting a 25-to 100-fold higher number of t and b lymphocytes, causing them to acquire surface il35. this surface il35 is absent when ev/exosome production was inhibited, or if ebi3 is genetically deleted in treg cells. summary/conclusion: the unique ability of ev to coat bystander lymphocytes with il35, promoting exhaustion in, and secondary suppression by, non-treg cells, identifies a novel mechanism of infectious tolerance. funding: nih grants r01-ai119140-03 (to w.j.b.), r01 ca203689 and p30 ca047904 (to d.a.a.v.) and the university of wisconsin carbone cancer center support grant p30 ca014520. unique formulated dual targeting antigen specific and delivered mirna-150 gene regulating exosomes acting at the immune synapse to induce apc-derived secondary suppressive exosomes introduction: an exosome-apc circuit we uncovered may be applicable beyond skin immunity we study in mice. methods: high antigen dose tolerized cd8 + t cells make suppressive antigen-specific exosomes due to chosen surface antibody light chains that enable targeting antigen presenting cells (apc) antigen-specifically for delivery of also chosen inhibitory mirna-150 to mediate specific functional gene alterations. results: both antigen and gene specificity aspects are lent to naïve but activated exosomes by simple in vitro incubations alone. for mechanism, these primary exosomes bind antigen peptides in mhc on apc that in turn make secondary suppressive exosomes that act peptide/mhc-specifically on the effector t cells at the immune synapse. they transfer another mirna for strong prolonged inhibition of active delayed-type hypersensitivity (dth) for days even, when the primary mirna-150-pos exosomes are administered orally at the height of the in vivo response, in a physiological dose. summary/conclusion: it is shown possible to induce therapeutic exosomes with ag targeting of choice due to placed ab on the surface and that also target specific gene functions of acceptor cells due to carriage of a selected mirna. this dual ag and gene-specific therapy has applications in treatment of cancer, autoimmunity and allergies. introduction: previously, our group characterized distinct populations of extracellular vesicle (ev) released from neutrophilic granulocytes: ev formed spontaneously (sev) and upon activation with opsonized particles (aev). the aev differs in protein cargo and its ability to inhibit bacterial growth. we described that mac-1 integrin (cr3 receptor) plays key role in the aev production and extracellular calcium supply is crucial in this signalization. in the present work, our aim was to investigate whether mac-1 activation or casignalling on their own are sufficient for the initiation of the aev biogeneis. methods: we isolated neutrophil derived evs from peripheral human blood and murine bone marrow by two-step centrifugation and filtration. we tested the effect of ca-ionophore and examined the ev production on c3bi coated surface and in soluble form. we quantified the vesicles by flow cytometry and determined their protein content by bradford assay. we examined their antibacterial effect in parallel with optical density-based measurement and our flow cytometry based method. results: on c3bi coated surface, we observed an increased ev production, and these evs possessed antibacterial capacity. however, in soluble condition, c3bi did not induce further ev production, and these evs did not show any antibacterial property. we found that ca-ionophore initiated ev formation, but these ev did not show antibacterial effect. we observed ev production increase after ca-ionofore treatment both in the presence and in the absence of extracellular ca. the ca-ionophore slightly increased the opsonized particle induced ev production, but did not potentiate their antibacterial capacity. summary/conclusion: mac-1 activation is not just crucial, but sufficient in initiation of the aev biogenesis. clustering of this receptor is required. while the ca-signal is crucial, it is not sufficient in the generation of aevs. extracellular vesicles and their microrna cargo in retinal health and degeneration: mediators of homoeostasis, and immune modulation yvette s. m. wooff, adrian cioanca, riemke aggio-bruce, joshua chu-tan, ulrike schumann and riccardo natoli the australian national university, canberra, australia introduction: photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases such as age-related macular degeneration (amd). however, the molecular mechanisms regulating these biological processes are largely unknown. extracellular vesicles (ev) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. evs, including exosomes, encapsulate and transfer microrna (mirna) to recipient cells and in this way can modulate the environment of recipient cells. dysregulation of evs however is correlated to a loss of cellular homoeostasis and increased inflammation. in this work we investigated the role of isolated retinal small-medium sized ev (s-mev) in the regulation of homoeostasis and immune modulation in both the healthy and degenerating retina. methods: isolated s-mev from healthy and degenerative (photo-oxidative damaged) mouse retinas were characterized using dynamic light scattering, transmission electron microscopy and western blot, and quantified using nanotracking analysis. small rna-seq was used to characterize the mirna cargo of retinal s-mev isolated from healthy and degenerating retinas. finally, the effect of exosome inhibition on s-mev-mediated immune modulation was investigated using systemic daily administration of exosome inhibitor gw4869 and analysed by in situ hybridization of s-mev-abundant mirna. electroretinography and immunohistochemistry were performed to assess functional and morphological changes to the retina as a result of exosome depletion. results: our results demonstrated an inverse correlation between s-mev concentration and photoreceptor survival, with decreased s-mev numbers following retinal degeneration. small rna-seq revealed that s-mevs contained uniquely enriched mirnas, however no differential composition in s-mev mirna cargo following photo-oxidative damage was observed. exosome inhibition using gw4869 exacerbated photoreceptor degeneration, with reduced retinal function and increased levels of inflammation and cell death seen following photo-oxidative damage. further, reduced translocation of the photoreceptor-derived s-mev was demonstrated following exosome-inhibition in photo-oxidative damaged mice. summary/conclusion: taken together, we propose that retinal s-mev and their mirna cargo play an essential role in maintaining retinal homoeostasis through immune-modulation, and have the potential to be targeted using gene therapy for retinal degenerative diseases. impacts of agricultural dust exposure on human lung-resident mesenchymal stromal/stem cells and their extracellular vesicles introduction: agricultural dust is considered a high-risk occupational hazard by the cdc, with impacts reaching throughout the communities surrounding these industries, leading to increased incidence of respiratory illness and disease among individuals within this occupation and these communities. lung-resident mesenchymal stromal/stem cells (lr-msc) have an important role in maintaining homoeostasis in the lung, and mediating pro-and anti-inflammatory effects, particularly during exposure to inhaled irritants, like agricultural dust. one way in which these lr-msc promote lung homoeostasis is through the release of extracellular vesicles (ev), with a variety of cargo that elicit changes among target cells. we hypothesize that exposure to agricultural dust modifies the quantity and cargo of ev released by lr-msc to promote lung tissue homoeostasis. methods: primary human lung-resident mesenchymal stromal cells were exposed to extracts of dusts collected from swine confinement facilities (de) for 8 or 48hrs and the media from these exposures were collected and enriched for ev by opti-prep density gradient ultracentrifugation. the quantity of these ev were assessed by nanoparticle tracking analysis. additionally, cytokine and chemokine release by lr-msc were analysed by enzyme-linked immuno assays. results: as assessed at 48 hr following treatment, deexposed lr-msc released pro-inflammatory cytokines, il-6 and il-8, with il-8 release reaching statistical significance at 0.1%, 0.5%, and 1% de concentrations (p = 0.0367, <0.0001, and <0.0001 respectively) and il-6 trending a similar dose response but only statistically significant at 1% de (p = <0.0001). de exposure of lr-msc also induced changes in the lr-msc-derived ev populations when compared to vehicle control, where lr-msc released significantly more ev in the 5 and 10% iodixanol fractions (p = <0.0001 and 0.0219, respectively) at 8 hr following de treatment. alternatively, there were significantly less ev in the 20 and 40% density fractions in the media of deexposed lr-msc versus vehicle control. summary/conclusion: following exposure to agricultural dusts, lr-msc-derived ev populations more likely consist of exosomes and ectosomes, which play an important role in promoting lung tissue homoeostasis during exposure-related pulmonary inflammation. introduction: during analyses of single extracellular vesicles (evs) by flow cytometry (fcm), particles below the detection limit may exceed the trigger threshold, which is called swarm detection and generates false-positive counts. serial dilutions are recommended to find the minimal dilution for which swarm detection is absent. however, because particle concentrations in plasma vary, the optimal dilution differs >100-fold between donors, but it is unfeasible to do serial dilutions for each clinical sample. therefore, our aims are to (1) develop a faster method to avoid swarm detection, and (2) increase the number of detected evs per second. methods: we measured serial dilutions of cd61stained evs in platelet free plasma (pfp), with and without spiking of fitc beads, by fcm (apogee a60-micro). we triggered either on side scatter or fluorescence. results: for scatter triggering with our fcm, swarm detection consistently occurred for plasma samples exceeding a (total particle) count rate of 4,100-4,200 events/s. the cd61+ evs concentration scaled linearly over 1.5 orders of magnitude of the dilution and most donors required >1000-fold dilution to avoid swarm detection, thereby reducing cd61+ ev counts. for fluorescence triggering, the cd61+ evs concentration scaled linearly over >3 orders of magnitude of the dilution. for all donors, swarm detection was absent after 32-fold dilution (relative to pure plasma). the count rates of cd61+ evs were 30-100-fold higher compared to scatter triggering. the spiked fitc beads confirmed that the median signals remained constant. summary/conclusion: we have developed two clinically applicable ways to avoid swarm detection. for scatter triggering, the count rate provides direct feedback on the presence of swarm detection in plasma samples. for fluorescence triggering, swarm detection was absent for all plasma samples diluted ≥32-fold and compared to scatter triggering, count rates of cd61 + evs were 30-100 fold higher, thereby improving statistical significance. funding: edwin van der pol is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni 15924. benchmarking flow cytometric analysis of nanoparticles: a cross-platform study for single extracellular vesicle detection introduction: despite flow cytometry being widely used to analyse cells in suspension, most commercial instruments lack sensitivity when measuring nanoparticles (nps) and extracellular vesicles (evs). furthermore, the use of appropriate reference materials (rms) for calibration and quality control are essential to compare results acquired with different instruments. to work towards successful clinical applications for ev biomarker profiling, benchmarking studies including state-of-the-art flow cytometers are required. we here investigated the ability of three different flow cytometers to detect nps and evs. methods: the instrument sensitivity of light scattering detection was evaluated by using synthetic nps of different sizes and refractive indices. fluorescent calibration was investigated by using molecules of equivalent soluble fluorophores (mesf) beads. biological recombinant evs (revs) were used to validate the detection and quantification of fluorescent evs in a side-by-side cross-platform study using an n30 nanoflow analyser (nanofcm), an optimized bd influx and a cytoflex lx. results: we found that when light scatter based detection was used, the nanofcm detected the smallest non-fluorescent nps, the bd influx was able to provide reliable fsc information from the smallest detected nps and the cytoflex performance was greatly improved by the use of violet-ssc. biological revs showed that the nanofcm could clearly resolve fluorescent evs while the bd influx and cytoflex were unable to fully resolve revs from background, although fluorescence threshold improved detection. in addition, our findings revealed that different concentrations are required to ensure single ev detection in these platforms. summary/conclusion: we identified several strengths and limitations for each platform with respect to single ev analysis. furthermore, our results showed that proper calibration and rms are of utmost importance to ensure reliable interpretation of ev flow cytometric data. caution when using membrane dyes for sequential extracellular vesicle analysis diana pham, michael wong, desmond pink and john lewis nanostics, edmonton, canada introduction: confirmation that particles detected by microflow cytometry are actually extracellular vesicles (evs), or at least membranous in composition, can be achieved through a variety of methods. positively staining particles with a membrane dye strongly suggest that the particle contains a membrane; loss of stain (or detection) after detergent solubilization of the membrane-dyed particles provides even stronger evidence that the particles were evs. it is important to recognize that the labelling protocol provided by the membrane dye manufacturer may not be ideal for all types of evcontaining biological samples, such as blood, urine, semen etc.). removal of excess dye from stained evs is very difficult and can be impractical depending on the nature of the experiment. however, this means that the potential for excess dye to contaminate subsequent sampling is high. therefore, it is important to determine optimal working concentrations and labelling conditions when using membrane dyes for ev detection to understand properties that may impact your analyses. methods: to assess the utility of membrane dyes, titration curves were generated to determine the optimal working concentrations of membrane dyes for ev detection in conditioned media and human serum samples. once the optimal concentration was determined the potential of dye carry-over from sample to sample during microflow cytometry detection was evaluated by tracking dye positive (dye+) particles in phosphate buffered saline (pbs) blanks and matched, unlabelled, sample replicates. results: we found that optimal concentration of any membrane dye is dependent on sample type. even with the inclusion of system washes to prevent sample carryover, there was carryover of low amounts of dye+ particles into sequentially analysed pbs blanks. if unstained samples were analysed following a stained sample, excess dye (or at least dye+ events) appeared in the data. a sample concentration effect was also seen; samples of lower concentrations were more susceptible to dye carryover. summary/conclusion: when using membrane dyes to stain evs in biological samples, especially if an autosampler is employed to run a series of tests, it is critical to determine the optimal concentration of dye for each type of sample, as excess dye can carry over to the next sample in the queue. in addition, determining the necessary steps to clean any excess dye following each sample run will improve the accuracy of ev detection and analyses. funding: nanostics alberta innovates alberta cancer foundation correlation between size and protein expression of single exosomes by combined atomic force and fluorescence microscopy introduction: there are no universal markers of extracellular vesicles, but often they are identified by the presence of tetraspanins in their membrane. based on this, products have been developed to precipitate or quantify evs by acting upon cd9, cd81, and cd63. however, evs also carry proteins from their parent cells, and capturing evs based their presence allows for a more complete understanding of vesicle heterogeneity from a single cell type, and for evs derived from specific tissues to be enriched from other biofluids in support of biomarker assessment. for example, evs derived from the brain could be captured from the general population of serum evs for better assessment of cargo associated with proteinopathy. the goal of this study was to identify specific antibodies to capture and label evs bearing the neural markers cd171, snap25, α-synuclein, tau, and ncam. methods: the targets were overexpressed in hek293 t cells through transient transfection of plasmids (origene). media was conditioned for 24-48 hours, and then centrifuged to remove cell debris. cell lysates and concentrated conditioned media (cm) were analysed by western blot. unpurified cm, or cm after performing size exclusion chromatography (sec, izon), were analysed in the exoview r100 system. diluted cm was incubated on custom antibody microarray chips overnight. then the chips were labelled with a cocktail of 3 labelled antibodies, washed and imaged. vesicles were counted, sized, and phenotyped. next, commercially available pooled human csf was analysed in a similar fashion to determine their abundance in a relevant biofluid. results: multiple antibody clones were tested in different combinations for capture and labelling for the five different neuronal enriched proteins of interest, and optimal combinations were identified. some markers were identified on particles > 50 nm in size that were negative for tetraspanins, while others colocalized with tetraspanins. through comparing permeabilized and intact evs with and without sec to remove non-vesicular proteins, we found that tau could be on the vesicle surface, within the vesicle, and free in solution. summary/conclusion: the exoview platform can be customized to enable the detection of proteins of interest and to determine whether they are on the ev surface, intravesicular, or non-ev associated. methods: forty non-smoking male and female subjects (40-70y) at moderate risk for cvd were recruited for the study. evs from platelet-free plasma (pfp) were isolated using size exclusion chromatography (sec). the concentration and size distribution of evs were measured by nanoparticle tracking analysis (nta) and flow cytometry (fcm). three ev markers, including annexin v for the circulating phosphatidylserinepositive (ps+) evs, cd41 for platelet-derived evs and cd105 for endothelial-derived evs were used for phenotyping. in addition, coagulation and fibrinolysis were assessed using a thrombodynamics analyser (hemacore). platelet aggregation to determine platelet function was assessed by a high-throughput platelet function assay with a wide range of concentrations of agonists, including adenine diphosphate (adp), collagen-related peptides (crp-xl), epinephrine, thrombin receptor activating peptide (trap-6) and u46619. the association between thrombogenic risk markers for cvd and ev numbers was tested by pearson's correlation coefficient and linear regression model using the statistical program, spss. results: circulating ev concentration with threshold of 71 nm, measured by nta, were positively associated with coagulation-related risk markers, including rate of clot growth (r = 0.568; p = 0.01) and clot size at 30 min (r = 0.480; p = 0.002). ps+ evs derived from endothelial cells, determined by fcm, were negatively associated with lysis onset time (r = −0.410; p = 0.009), whereas they were found positively correlated with lysis progression (r = 0.417; p = 0.007). both mean and mode size of cevs, detected by nta, were significantly correlated with u46619-induced platelet aggregation (r = −0.338; p = 0.033, r = −0.382; p = 0.015, respectively). summary/conclusion: in subjects at moderate risk for cvd, cev numbers were positively related to rate of clot growth and clot size and size of cevs was negatively related to platelet activity. higher numbers of endothelial cell-derived ps+ cevs were associated with lower rates of fibrinolysis. this suggests that cevs promote clot growth and reduce fibrinolysis, and may therefore be an indicator for greater risk of cvd. beyond stem cells: extracellular vesicles from human induced pluripotent stem cells (hipsc) and hipsc-cardiomyocytes as therapeutic approaches for heart failure introduction: heart failure is caused by a variety of underlying diseases, the most common being myocardial infarction. initially regarded as an alternative to pharmacological approaches, stem cell transplantation has failed to demonstrate clinically meaningful results. instead, it has become increasingly apparent that the therapeutic effects of transplanted cells are largely mediated by their secretome, while mounting evidence suggests extracellular vesicles (evs) play a major role in cardiac repair. within this framework, evs from human induced pluripotent stem cells (hipsc) and hipsc-derived cardiomyocytes (hipsc-cm), hold a tremendous potential to treat cardiovascular disease. we isolated evs from conditioned culture media at key stages of the hipsc-cm differentiation and maturation processes, i.e. from hipsc (hipsc-ev), cardiac progenitors (cpc-ev), immature (cmi-ev) and mature (cmm-ev) cardiomyocytes, with the aim of studying their potential role as therapeutics, and whether their effectiveness was influenced by the state of their parent cell. methods: hipsc were differentiated into cardiomyocytes in a 3d culture approach, using the protocols developed by our group. ev isolation was performed on an iodixanol density gradient, and the evs were characterized in terms of particle size and particle size distribution, presence of ev-specific markers, and imaging through transmission electron microscopy. functional studies were performed using human umbilical vein endothelial cells (huvecs) to evaluate evuptake, cell migration and angiogenesis. results: evs from all hipsc and cardiac derivatives presented a typical cup-shaped morphology and expressed cd63 and cd81. ev yield varied along differentiation, with a minimum for cpc and a maximum for cmi. pkh26-labelled evs were uptake by huvecs, and colocalized with calnexin, a protein from the endoplasmic reticulum. wound healing assays showed an increased cell migration in huvecs treated with cardiomyocyte-derived evs, in comparison with control evs isolated from foetal bovine serum. summary/conclusion: our findings suggest a different ev secretion profile along cm differentiation and maturation, with preliminary assays showing ev functionality. ongoing work aims at elucidating the possible differences in function and cargo amongst these types of evs. endothelial cells differentially load and secrete extracellular vesiclederived micrornas into apical and basolateral compartments this may play a role in microcalcification in calcific aortic valve disease (cavd), but this is poorly understood. annexin a1 is thought to be a marker of membrane-derived evs, but because it can be found on the cytoplasmic or extracellular side of the plasma membrane, its localization within or on the surface of evs is unclear. the goal of this study was to determine whether annexin a1 is found on the surface of evs in two cell lines relevant to cavd, and develop an assay that can be used to determine whether this changes under pathogenic conditions. methods: evs were isolated by differential ultracentrifugation from the conditioned medium (cm) of smooth muscle cells (smc) and valvular interstitial cells (vic). total protein in the cell lysates and ev pellets was analysed by western blot. evs from cells treated with control sirna or anxa1-sirna were enumerated and phenotyped using the exoview r100 platform. evs with surface expression of cd9, cd81, cd63, and annexin a1 were captured using a customized antibody microarray chip. then evs were labelled with fluorescent antibodies to assess ev number, size, and colocalization of ev proteins. the knockdown of annexin a1 allowed us to assess the specificity of the selected annexin a1 antibody. results: the ev fraction was positive for cd9, and lacked markers of other vesicle types. western blot on the ev pellet and supernatant in ± edta indicated that there is annexin a1 both on the surface of and within the evs. using the antibody microarray chips, numerous annexin a1+ evs were captured on the annexin a1 spots from the control cm, and there was a marked decrease in capture and labelling from anxa1-sirna treated cells. under both conditions, vesicles were also captured on tetraspanin probes, with the greatest number captured on cd9, then cd63 and cd81. there was a significant population of annexin a1+ evs that was negative for tetraspanins. summary/conclusion: annexin a1 is found on the surface of evs. the assay developed in collaboration with nanoview biosciences is well suited for assessing the number and phenotype of annexin a1+ evs derived from smc and vic cell lines, which could provide a useful method for understanding ev populations in cavd patient cell lines. funding: this work was supported by hl136432 and hl147095. possibility of exosomal micrornas associated with chronic limb-threatening ischaemia, the end stage of atherosclerosis, as a promising biomarker introduction: chronic limb-threatening ischaemia (clti), the end stage of peripheral artery disease (pad), has poor prognosis and is attributed to lifestyle disease. with increasing of atherosclerotic disease all over the world, establishment of biomarker for should play a pivotal role for early detection and preventing aggravation of the disease. the aim of this study is to explore the possibility of liquid biopsy for atherosclerotic disease by analysis of clti-associated exosomal micrornas. methods: clti due to pad was diagnosed by anklebrachial blood pressure index, skin perfusion pressure (<40 mmhg) and angiography. ten preoperative clti patients and 10 control patients without pad were analysed (all patients with diabetes and 50% of patients had end-stage renal failure [esrd] ). to identify biomarkers associated with clti, exosomes were extracted from patient's serum after ultracentrifugation and total rna including small rna was isolated from the exosomes. the expression profile of exosomal micrornas associated with clti were evaluated using a next generation sequencing. results: forty-three exosomal mirnas associated with clti were identified. intriguingly, these mirnas were clearly categorized with esrd, which was well known as end-stage of life-style disease: these were stratified into 20 micrornas for esrd patients and 23 micrornas for non-esrd patients. since esrd is the most important factor significantly related to patient's prognosis in clti, exosomal micrornas reflected patient's comorbidity onto the expression profile. summary/conclusion: a portion of the expression profile of exosomal micrornas associated with clti was identified. exosomal microrna could be a biomarker to stratify patient's condition along with their comorbidities and is very promising for individualized diagnosis in atherosclerotic diseases with risk diversity. postoperative plasma exosomal mir-21 and mir-133a signature in patients with left ventricular reverse remodelling after surgical mitral valve repair underwent implantation of a prosthetic mitral ring. lv remodelling was assessed by cardiac magnetic resonance imaging and pexos were isolated by optimized ultracentrifugation before surgery (t0) and six months after surgery (t1). isolated pexos were quantified by nanoparticle tracking analysis and mir-1, mir-21, mir-133a, and mir-208a were measured by rt-qpcr. the same analysis was performed on healthy subjects with normal cardiac function (n = 8). local ethical committee approved the study (emigrate study, approval n°1529) and informed consent was obtained from all patients. results: pexos levels at t0 were lower (−32%, p = 0.02) in patients with worst postoperative lv function, while they were higher at t1 (+31%, p = 0.03) in patients with reversed lv remodelling after surgery. at t1, the increase in pexos levels was associated to decreased heart mass index (−13%, p = 0.02) and higher levels of exosomal mir-21 (+78%, p = 0.02) and mir-133a (+69%, p = 0.05) were detected in patients with improved lv function. summary/conclusion: higher postoperative levels of pexos delivering mir-21 and 133a depict lv reverse remodelling after surgical mitral valve repair. monitoring of exosomal micrornas cargo might predict postoperative outcome in patients with mr. expression of lipocalin-2 (lcn2) in circulating extracellular vesicles (evs) and femoral plaque-derived evs of peripheral arterial disease patients. introduction: clinically, the drug resistance situation of acinetobacter baumannii is becoming increasingly serious, and its drug resistance has become a difficult problem for nosocomial infection and clinical treatment. in view of the relatively slow development of antibacterial drugs, exploring the resistance mechanism of acinetobacter baumannii is of great significance to improve bacterial resistance and help clinical treatment. studies have shown that outer membrane vesicles (omvs) can transmit resistance genes to mediate the spread of drug resistance, and recent studies have confirmed that high expression of efflux pumps play an important role in the multidrug resistance of a. baumannii. in this study, we want to explore whether the outer membrane vesicles of acinetobacter baumannii can transfer the efflux pump related substances. methods: first, ultracentrifugation and density gradient centrifugation were used to extract the omvs of acinetobacter baumannii antimicrobial-sensitive strains (atcc19606) and antimicrobial-resistant strains. then, nanoparticle tracking analysis (nta) technology was used to analyse the particle size and distribution range of omvs. transmission electron microscopy (tem) was used to identify their morphology and structure. bradford method was used to determine the protein concentration of omvs. next, the omvs of antimicrobial-resistant strains were incubated with the antimicrobial-sensitive strains and then the drug susceptibility test was done to determine whether omvs of antimicrobial-resistant strains could transmit antimicrobial-resistance information to the antimicrobial-sensitive strains. finally, pcr, qpcr and mass spectrometry were used to determine whether the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. results: nanoparticle tracking analysis (nta) detected the concentration and size distribution of omvs of acinetobacter baumannii strains. it showed that the extracted omvs have a relatively uniform particle size and a size between 100-250 nm. tem showed that omvs had a typical vesicle structure. omvs coculture experiments showed that omvs of the antimicrobial-resistant strains can indeed pass resistance to the antimicrobial-sensitive strains. and the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. summary/conclusion: omvs of the antimicrobialresistant strains can indeed pass resistance to the antimicrobial-sensitive strains. the cause of acquiring antimicrobial resistance in sensitive strains may be caused by resistant strains passing efflux pump-related genes or proteins to sensitive strains. characterization of melanocytic extracellular vesicles during ageing of the choroid kelly coutant a , léo piquet a , nathan schoonjans b , philippe gros-louis a , julie bérubé c , stéphanie proulx a , alain r. brisson d and solange landreville a a université laval, quebec city, canada; b université de lille, lille, france; c centre de recherche du chu de québec-université laval, quebec city, canada; d université de bordeaux, bordeaux, france introduction: the choroid is located at the backside of the light-sensitive retina and is highly vascularized. it contains pigmented melanocytes, and their melanin protects them against oxidative stress. since ageing reduces the number of melanosomes in melanocytes and generates a stiffer extracellular environment, our hypothesis is that surrounding choroidal cells and the retinal pigment epithelium (rpe) are subject to more oxidative stress-related damages. this study aimed to characterize evs released by human choroidal melanocytes in the context of intercellular cooperation during ocular ageing. methods: melanocytic evs were recovered from the conditioned culture medium of young/old melanocytes grown on hydrogels of varying stiffness (0.5-20 kpa) by differential centrifugation. the concentration and size distribution of melanocytic evs were determined by high-sensitivity flow cytometry. cryo-transmission electron microscopy combined with receptor-specific gold labelling were used to reveal their morphology, size and phenotype. the relative abundance of 8 surface markers was evaluated with the exo-check exosome antibody array. the uptake of fluorescent melanocytic evs by the rpe and choroidal endothelial cells was assessed by confocal microscopy. results: choroidal melanocytes released evs positive for annexin-5 and the tetraspanin cd63. young melanocytes produced more annexin-5 positive evs and evs larger than 500 nm compared to older donors. the stromal stiffness impacted the concentration and size of melanocytic evs. we confirmed the uptake of melanocytic evs by endothelial and rpe cells. summary/conclusion: evs from choroidal melanocytes are internalized by surrounding endothelial cells and rpe. age-related stressors modify the phenotype of melanocytic evs. the identification of melanocytic factors that can protect retina/choroid cells from oxidative stress-induced cell death could lead to more efficient therapy for patients suffering from dry agerelated macular degeneration. introduction: owing to their proposed biocompatibility and ability to cross biological barriers, evs represent an attractive therapeutic delivery platform. however, evs are eminently heterogeneous. a better understanding of ev heterogeneity and its origins will allow for improved design of ev-based therapeutics. ev heterogeneity is mainly studied by focusing on distinct ev subpopulations. other sources of heterogeneity, such as heterogeneity within ev secreting cells themselves, have been investigated in lesser detail. in this study, we assessed the phenotypic drift of cell derived evs to explore the origins of ev heterogeneity and its potential impact. methods: three independent samples of two mda-mb-231 breast cancer cell sub-clones were cultured for six weeks. evs were harvested weekly and analysed using the macsplex exosome flow cytometry kit. at two time points the proteome of evs was analysed by lc-ms/ms mass spectrometry with subsequent gene ontology and reactome pathway analysis. results: the expression of over 600 proteins was deregulated in evs derived from the two different cell clones. many de-regulated proteins were associated with biological processes predicted to affect potential ev toxicity (platelet activation, neutrophil degranulation, blood coagulation) and ev biological activity (antigen presentation, inflammation, tgf-beta/ mtor/wnt signalling). more surprisingly, within only two weeks, over 400 ev proteins, many associated with immune modulation, apoptosis, interleukins, cytokines and cell signalling pathways (including those affecting t-cell/b-cell receptors) were de-regulated between the two ev isolation time points. summary/conclusion: results suggest that temporal changes can be observed in the ev proteome (potentially by clonal drift, epigenetic changes or cellular genomic instability) over short time periods. these changes could cause significant differences in biological effects and delivery capabilities between evs harvested from the same cells at different time points and conditions. in vivo tracking and biodistribution analysis of mesenchymal stem cellderived extracellular vesicles in a radiation injury murine model introduction: recent studies indicated that extracellular vesicles (evs) play key roles in intercellular communication and have great potential for clinical application. understanding the biodistribution of evs is therefore essential. our previous works have shown the ability of mesenchymal stem cell (msc)derived evs to protect haematopoietic cells from radiation damage. in this study, we evaluated the biodistribution of msc-evs in a radiated mouse model. methods: human msc-evs were harvested by ultracentrifugation and labelled with did lipid dye. the reliability of the labelling evs was confirmed by sucrose gradient fractionation analysis. the distribution of evs in radiation-exposed mice after ev intravenous administration were evaluated by fluorescence molecular tomography and further confirmed by flow cytometry and confocal microscopy analysis. results: we observed that did labelled msc-evs appeared highest in liver and spleen, lower in bone marrow in tibias, femurs, and spine, and were undetectable in heart, kidney and lung. we found the significantly increased msc-ev accumulation in spleen and bone marrow post-radiation appeared with an increase of uptake of msc-ev by cd11b+ and f4/80 + cells, but not b220+ cells, compared to those organs from non-irradiated mice. however, there was a predominant ev accumulation in lung and less accumulation in spleen and liver; in mice infused with human lung fibroblast cell derived evs (lfc-evs) and there was no significant lfc-evs accumulation change in the spleen or liver after radiation. we further found that increasing levels of irradiation caused a selective increase in vesicle homing to marrow and spleen. this accumulation of msc-evs at the site of injured bone marrow could be detected as early as 1 hour after msc-ev injection and was not significantly different between 2 and 24 hrs. post-msc-ev injection. summary/conclusion: this study indicated the specific accumulation of ms-evs at the site of injury of haematopoietic tissue in radiation injury mice. funding: this work was supported by the nih grants 5uh2tr000880, 3uh3tr000880-03 s1, 5p20gm11 9943, and 5t32hl116249. linking fat to colorectal cancer: extracellular vesicle crosstalk sheffield hallam university, sheffield, uk introduction: colorectal cancer is the third most common cancer worldwide, and fourth leading cause of malignancy related mortality. understanding the mechanisms of its growth and metastasis is key to elucidating new therapeutic targets and developing treatments in the clinical setting. epidemiological evidence indicates an increased risk of cancer in obese patients, pointing to bidirectional communication between colon and adipose cells. extracellular vesicles (evs) are small membrane enclosed packages released by cells, capable of transporting bioactive cargo from donor to recipient cells and inducing phenotypic changes. adipocytes are a key component of the tumour microenvironment and interactions between adipose tissue and tumour cells may be important in the growth and metastasis of cancer. in this study, we investigate the effects of colorectal cancer evs on adipocytes in vitro, and potential induction of dedifferentiation to a more fibroblastic, pro-inflammatory phenotype. methods: evs were isolated from sw480 and ht29 human colorectal cancer cell lines by differential ultracentrifugation and mature adipocytes generated by differentiation of the sgbs human pre-adipocyte cell line. adipocytes were treated with evs and their lipid content measured by oil red o to determine loss of lipids. inflammatory cytokine profile was measured by elisa to assess any increase in pro-inflammatory behaviour, and expression of late adipogenesis markers were determined by western blot. results: ev treatment was shown to reduce lipid accumulation in adipocytes, with up to 80% reduction in lipids observed at the 50 µg/ml dose. treatment was also shown to reduce the expression of late adipogenesis markers, and increase secreted levels of proinflammatory cytokines il-6 and il-8 by over 3 fold and 10 fold respectively. these results provide evidence for colorectal cancer derived ev involvement in the dedifferentiation observed in cancer associated adipocytes in vivo, displaying an altered phenotype, releasing lipid energy stores to fuel tumour growth and increasing pro-inflammatory signalling. summary/conclusion: studies have shown colorectal cancer evs may be involved in signalling which induces functional changes in cells within the tumour microenvironment. our work indicates that ev mediated dedifferentiation of resident adipocytes may potentially contribute to a microenvironment favouring cancer cell growth and metastasis. further work aims to elucidate the specific ev cargo which mediates these effects. introduction: ageing is a major risk factor for many human diseases. it is a complex process that progressively compromises most of the biological functions of the organisms, resulting in an increased susceptibility to disease and death. senescence is a cellular phenotype characterized by a stable cell cycle arrest. senescent cells are accumulated in the body during ageing. it contributes to develop age-related diseases and cancer. the alteration in intercellular communication with age has been demonstrated to be due to senescent cells developing a phenomenon denominated senescenceassociated secretory phenotype (sasp). exosomes are small extracellular vesicles (sev) (30-120 nm) of endocytic origin whereas microvesicles are formed by shedding of the plasma membrane. they contain nucleic acids, proteins and lipid that generally reflect the status of the parental cell and can influence the behaviour of neighbouring cells. methods: in this study, we demonstrated that the small extracellular vesicles (sev) contribute for transmitting paracrine senescence to proliferative cells firstly, we evaluated the presence of exosome-like particles in the sev from senescent cells by detection of exosome markers (alix, tsg101 and cd63), transmission electronic microscopy (tem) and nanoparticle tracking analysis (nta). to determine that sev from senescent cells are mediators of the paracrine senescence, we performed functional assays using cre-loxp reporter system and high-throughput results: besides, we confirmed at a single-cell level that the proliferative cells internalizing sev from senescent cells activate senescence process using the cre-reporter system. sev protein analysis from senescent cells by mass spectrometry (ms) and validation of top candidates using a functional sirna screen identify interferon induced transmembrane protein 3 (ifitm3), a component of non-canonical interferon (ifn) pathway, as partially responsible for transmitting senescence to proliferative cells. summary/conclusion: in conclusion, we found that sev are regulators of paracrine senescence and ifitm3 contained in senescent sev has an important role in the intercellular communication mediated through sev during cellular senescence1. bin wu a , lei guan a , ye xu a , likang chin a , ting li a , youhai chen a , gordon mills b , jinqi ren a , ravi radhakrishnan a , rebecca wells a and wei guo a a university of pennsylvania, philadelphia, usa; b oregon health & science university, portland, usa introduction: extracellular matrix (ecm) remodelling and stiffening are associated with solid tumour progression. stiff ecm promotes cell proliferation, epithelial-to-mesenchymal transition (emt), metastasis and chemoresistance. hepatocellular carcinoma (hcc) appears frequently in patients with liver cirrhosis or fibrosis while the mechanism remains unclear. exosomes have been determined to serve as messengers to mediate intercellular communication and influence the extracellular. tumour-derived exosomes have been shown to influence tumour progression, metastasis, drug resistance, angiogenesis and immune regulation. thus, determining whether exosomes provide a mechanism by which stiff matrix modulates tumour microenvironment for tumour progression opens a new way to understand cirrhosis and oncogenesis. here we identified the molecular mechanism of matrix stiffening induced exosome secretion and showed the different effect of exosomes induced by soft or stiff matrix on tumorigenesis. methods: huh7 cells were cultured on acrylamide gels with the stiffness was modulated to 500 pa (soft) or 10 k pa (stiff). the exosomes in conditioned media were collected and analysed by nanoparticle trafficking analysis (nta) and immunoblotting. protein expression level in cells was screened by reverse phase protein array (rppa). inhibitor or shrna were used to inhibit target proteins function. in vitro phosphorylation and gef assay were used to verify rabin8 phosphorylation and activation. exosomes from cells on soft or stiff matrix were injected into mice to study their effect on tumour growth. results: (1) stiff matrix promoted exosomes secretion. (2) akt was activated by stiff matrix and was required for exosome secretion. summary/conclusion: matrix stiffening promotes exosome secretion via akt-rabin8-rab8 pathway, contributing to tumorigenesis. tridimensional fibroblast culture revealed a novel exososome-dependent extracellular matrix secretion mechanism vincent clément a , bastien paré b , cassandra goulet a , thiéry de serres-bérard a , stéphane bolduc a , françois berthod a and françois gros-louis a a université laval, québec, canada; b norgen biotek corp., thorold, canada introduction: the extracellular matrix (ecm) is constituted of a variety of proteins and polysaccharides that are secreted locally and assembled into a thick 3d meshwork to provide biophysical and biochemical support to the surrounding cells, and regulate numerous cellular functions such as adhesion, migration and proliferation. dysregulation of ecm components or aberrant ecm remodelling can lead to various pathologies, as well as to play important roles in wound healing. although ecm secretion pathways are still largely unknown, the current paradigm is that ecmassociated proteins are synthesized in the endoplasmic reticulum and transported via the endosomes to the golgi apparatus en route to the cell surface and released by exocytosis. methods: to study ecm secretion pathway, we used 3dimensional (3d) cultured fibroblasts. this culture method technique has been used widely to generate tissue-engineered self-assembled stromal tissues, free of exogenous materials, and rely on long-term supplementation of sodium ascorbate into the culture medium. non-cancerous fibroblasts, grown in conventional two-dimensional (2d) cellular cultures, are known to be a poor source of secreted exosomes when compared to cancerous fibroblasts. results: here, we provide evidence that non-cancerous dermal fibroblasts can secrete high amounts of exosomes, containing different ecm proteins, when cultivated in a 3d fashion. we also demonstrated that dermal fibroblast-derived exosomes had the capacity to travel from one cell to another, induce cellular migration and promote wound healing. summary/conclusion: altogether, these findings reveal a novel exosome-dependent ecm deposition mechanism and suggest that the use of 3d-fibroblast cellular culture may emerge as an innovative approach in precision medicine to better study the role of patient-derived exosomes and ecm proteins in the establishment of cellular microenvironment in health and disease. anthony yan-tang. wu a , charles lai, yun-chieh sung b , steven t. chou c , vanessa guo c , jasper c. chien c , john j. ko c , alan l. yang c , ju-chen chuang c , hsi-chien huang b , syuan wu c , meng-ru ho d , maria ericsson e , wan-wan lin f , koji ueda g , yunching chen h , chantal hoi yin cheung i and hsueh-fen juan j introduction: bionanoparticles including extracellular vesicles and exomeres (collectively termed evs), have been shown to play significant roles in diseases and therapeutic applications. however, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a limited suitable method. methods: we developed a bioluminescence resonance energy transfer (bret)-based reporter, palmgret, to enable pan-bionanoparticle labelling ranging from exomeres (< 50 nm) to small (< 200 nm) and medium and large (> 200 nm) evs and larger evs (> 50 nm). results: palmgret emits robust, sustained signals and allows the visualization, tracking and quantification of bionanoparticles from whole-animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, bret, and fluorescence. using palmgret, we show that evs released by lung metastatic hepatocellular carcinoma (hcc) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. ev proteomics identified hcc-ev lung tropic protein candidates associated with cancer progression, in which slco2a1 and clic1 expression on non-tropic evs conferred lung-tropism, while cd13 gave spleen tropism. our results further demonstrate that redirected lung tropism decreases ev distribution to the liver, whereas the spleen tropism significantly reduces over time delivery to most major organs distribution including the liver and kidney. summary/conclusion: we established a multimodal and multi-resolution palmbret method to enable pan-bionanoparticle labelling and imaging and therefore quantification in live cells, whole animals, and preserved tissues. the method can resolve the intricate spatiotemporal dynamics of evs. palmgret revealed that evs derived from lung metastatic hcc are lung tropic, and the tropism can be conferred to non-lungtropic ev-293 t by decorating evs with identified hcc-ev membrane proteins. importantly, the enhanced ev delivery to tropic organs also significantly alters its distribution to other major organs. our findings suggest that the dynamics of ev biodistribution and targeted design should be investigated at the organ systems level in ev biology and therapeutic developments, respectively. tracking mesenchymal stem cell-derived extracellular vesicles (evs) in a in vivo cancer model introduction: small extracellular vesicles (sevs) are nanoparticles (30-120mn) encircled by a phospholipid bilayer, derived from the endocytic pathway and released by all cells. sevs have an inherent role in cell communication and deliver cargo to target cells. mesenchymal stem cells (mscs) and have a natural ability to home to tumours and metastases while avoiding the host immune response. it is hypothesised that msc derived sevs (msc-sevs) also possess tumourhoming and immune-evading capacities therefore could provide a novel targeted delivery vehicle for treatment of cancer. it is imperative to elucidate msc-sevs migratory itinerary in vivo to support translation to the clinical setting. methods: this study aimed to image the interaction of labelled msc-sevs with cancer cells in real time in vivo. sevs were isolated from wildtype mscs and mscs with stably expressing red fluorescent protein (rfp) (via lentivirus) by the combined techniques of differential centrifugation, microfiltration and ultracentrifugation. isolated sevs were extensively characterised by transmission electron microscopy (tem), nanoparticle tracking analysis and western blot. nod scid gamma (nsg) mice with dorsal skinfold window chamber (dsfwc) were injected with either mda-mb-231 luciferase (luc) expressing cells or ht-29-luc cells. bioluminescence imaging was performed to confirm tumour formation. a dose of 1x10^7 msc-rfp-sevs was directly added to the window chamber and rfp expression detected using a microscope with rfp filter attachments. 8x10^7 evs were incubated with the radionuclide, technetium-99m tagged duramycin (99 mtc-dur) for 30 minutes at room temperature. excess radiolabel was removed using exosome spin column (invitrogen™). the 99 mtc-dur-sevs were then added directly to the window chamber and charged particle imaging carried out. results: 18 hours post-administration; the rfp signal was localised at the tumour site. radiolabelled sev signal could be detected 10 minutes and 4 hours after administration. msc-sevs were successfully detected at the tumour site following direct administration using two different tagging and imaging approaches. summary/conclusion: this promising preliminary data supports the potential of this approach for tracking msc-sev migration in vivo. future studies will investigate systemic tracking of msc-sev migration. vaughn garcia 1 ; aejez sayeed 2 ; rachel derita 1 ; shiv ram krishn 3 ; peter a. introduction: tumor-derived small extracellular vesicles (sevs) have emerged recently as mediators of tumorigenesis. however, the role of sevs in response to irradiation, a widely used therapy in prostate cancer, is not fully understood. methods: our study involved the tramp mouse model of prostate cancer. we used plasma sevs isolated using differential ultra-centrifugation and further isolated using iodixanol gradient fractionation. we also used nanoparticle tracking analysis (nta) to analyze sevs. mouse pelvises were irradiated using 10 gy, for 5 consecutive days. results: we first observed that upon pelvic irradiation of tramp mice, the levels of the signaling oncogene c-src are reduced in plasma-derived sevs, while the average size of sevs is increased from 50-100nms to 70-250nms. furthermore, we show that the sevs from irradiated cells lose the ability to stimulate anchorage independent growth and migration of recipient cancer cells. additionally, sevs from irradiated mice increase the amount of dna damage in recipient cancer cells. summary/conclusion: overall, our data show that irradiation of tramp mice (and prostate cancer cells) significantly reduces the pro-metastatic and pro-anchorage-independent growth potential of sevs when tested on human cells. changes to the composition and behavior of a cancer cell sev population via radiation therapy offers promise for future therapeutic approaches for prostate cancer. introduction: there are emerging physiological and pathological functions of extracellular vesicles (evs) in neurodegenerative diseases including alzheimer's disease (ad). brain derived-evs contain pathogenic proteins, such as tau, amyloid beta (aβ), which have been reported to contribute to cell-to-cell propagation in those diseases. investigation of the brain-derived ev cargo, therefore, is important to further understand the mechanisms of progression in neurodegenerative diseases. we developed the ev separation method from unfixed frozen mouse and human brain tissues and assessed the protein composition. methods: to establish the ev separation method, we separated evs from frozen mouse brain tissue using sucrose density gradient ultracentrifugation (sg-uc) or size exclusion chromatography to compare the results from the particle number, morphology and protein profiling by nta, tem and mass spectrometry. evs were then separated from cortical grey matter of ad (n = 20) and control (n = 18) by sg-uc. tau and aβ in the evs were measured by immunoassay. differentially expressed ev proteins were observed by quantitative proteomics employing machine learning. results: the separated evs were enriched in ev molecules and devoid of contaminant proteins by sg-uc, showing our method was successful. the levels of ps396 tau and aβ1-42 were significantly increased in ad evs. annexin a5 (anxa5), neurosecretory protein vgf, neuronal membrane glycoprotein m6-a (gpm6a), and alpha-centractin (actz) were differentially expressed in ad evs. a combination of these 4 proteins were confirmed to predict ad with the 88% accuracy by machine learning. summary/conclusion: these data suggest our method were suitable for the separation of brain-derived evs and ev anxa5, vgf, gpm6a and actz can be potential biomarkers for monitoring the progression of ad. edta stabilizes the concentrations of extracellular vesicles during blood collection introduction: to establish reliable biorepositories for research on extracellular vesicles (evs) as disease biomarkers, the release of evs during blood collection and handling must be avoided. currently, citrate is recommended as the anticoagulant for blood ev research, but citrate does not inhibit the release of evs from activated platelets. the release of platelet-derived evs excludes pneumatic tube transport and makes assays time dependent, thereby limiting clinical compatibility. therefore, we aim to stabilize the release of platelet ev concentrations. methods: blood samples were collected from healthy individuals and subjected to common circumstances known to induce platelet activation. blood was (i) incubated with or without thrombin receptor-activating peptide 6 (trap; n = 7), a potent platelet activator, (ii) send to the lab by a routine blood transport (pneumatic tube system; n = 3), and (iii) stored at room temperature or at 4°c for 6 hours (n = 3). the concentrations of evs from platelets (cd61+), activated platelets (p-selectin+), erythrocytes (cd235a+), and leukocytes (cd45+) were determined by flow cytometry (apogee a60-micro). results: following activation by trap, concentrations of platelet-derived and activated platelet-derived evs increased 5.8-fold and 10.4-fold in citrate-anticoagulated blood, compared to 1.4-fold and 2.0-fold in edta-anticoagulated blood (edta vs citrate: p = 0.018 and p = 0.043, respectively). preliminary data show that during pneumatic tube transport and routine sample handling, both platelet-and activated platelet-derived evs were more stable in edta compared to citrate. the concentrations of evs from erythrocytes and leukocytes were unaffected under all studied conditions. summary/conclusion: to conclude, edta stabilizes platelet ev concentrations during and after blood collection, which would facilitate pneumatic tube transport, enhance reliability and thereby improves the establishment of reliable biorepositories for ev research. introduction: cancer-cell secreted extracellular vesicles, called exosomes, are an emerging biomarker for cancer liquid biopsy. profiling of cancer-associated exosomes usually required lengthy, and multi-step procedures; therefore simple and easy-setup sensing methods are urgently needed for diagnosing cancer in a timely manner. chirality, the foundational property of all biomolecules, including exosomal proteins, can be utilized for exosome detection and differentiation using recent advances in chiral nanostructures. we found that microfluidic sensors can be successfully implemented for successful detection of cancer-associated exosomes taking advantage of unusually high circular dichroism (cd) of chiral gold nanoparticles (aunps). circular dichroism-based exosome (cdexo) detection utilizes chiroplasmonic enhancement of cd signatures of cancer-associated exosomes. we first synthesized donut-shaped aunps conjugated with l-cysteine and immobilized the aunps on a glass slide using a layer-by-layer assembly. the aunps on slide glass were surface functionalized by the standard biotin-avidin reaction after mua treatment. biotinylated annexin v marker, targeting phosphatidylserine (ps) expression on cancer-associated exosomes, was conjugated to the aunp surface. 5 μl of exosome samples from cancer cells (a549 and h3255) or normal cells (mrc5) were injected into the pdms microfluidic device and incubated for 30 minutes. the cd signal before and after exosome exposure was monitored, compared, and systematically analysed as a rapid technique for the detection of exosomes with high sensitivity. results: we showed that the cdexo signals from cancer exosomes showed 26.6 folds absolute cd peak value change and 3.41 folds shift, respectively, compared to that of healthy exosomes. importantly, the cdexo sensing method takes less than 10 mins in terms of total scanning time and requires minimal sample volumes. from the preclinical studies using 7 blood samples from cancer patients and healthy donors, we found that cancer patients show stronger band shift and signal change comparing to that of healthy donors, implying our platform could be used for cancer diagnosis. summary/conclusion: this new versatile and sensitive method based on chiroplasmonic exosome detection paves the way to profiling disease-associated exosomes in a timely manner for minimal volumes of liquid biopsies. ev classification and fractionation strategy using surface charge labelling takanori ichiki a , hiroaki takehara a , hirofumi shiono b and hiromi kuramochi a a the university of tokyo, bunkyo, japan; b innovation center of nanomedicine, bunkyo, japan introduction: the development of new classification technology is required based on the evaluation of physicochemical properties of exosome surfaces and the diversity of constituent molecules. in this presentation, we present the electric charge activated exosome sorting platform comprising microfluidic device technology and electric charge labelling technique. methods: the single nanoparticle analysis platform, which has been developed by our research group, images rayleigh scattered light (elastically scattered light) obtained by irradiating nanoparticles with convergent laser light and provides information of individual particles by image processing. the method that utilizes electrokinetic phenomena, unlike the method using fluorescent labels, measures the properties of the particle surface without serious difficulty in principle even if the particle size is on the order of tens nanometres, and further enables to perform fractionation. since the number of particles usually handled in exosome research or its envisioned application is enormous, it is not realistic to take an approach such as a cell sorter in which particles are sequentially manipulated one by one following the measurement results of individual particles. results: particles receive attraction or repulsion by an external field according to the charge density on the surface, so there is no need to control the external force, and it is possible to design a device that can autonomously fractionate particles according to the difference in zeta potential. summary/conclusion: in conclusion, we have proposed and demonstrated the new concept of electric charge activated ev sorter. funding: this research was partially supported by the center of innovation program (coi stream) from the japan science and technology agency. high throughput exosome analysis by using reversible microfluidic electrochemical sensor system introduction: exosome is one of the important extracellular vesicles (evs) released from parental cells and it contains various types of molecular cargos from its original cell including proteins, messenger rna (mrna), and micro rna (mirnas) [1] . the exosomes have recently emerged as biomarkers for early stage cancer detection because the number of exosomes originated from cancerous cells are significantly higher than those from normal cells [2] . since many different types of exosomes exist in the whole blood, it is necessary to isolate and detect disease-specific exosomes. for this reason, the isolation and the detection of exosomes is an important research issue and has been studied by many groups. however, limitations such as low throughput and low recovery still make it difficult to use exosomes in diagnostics and therapeutics. methods: in this study, we developed an integrated microfluidic electrochemical biosensor to extract plasma from whole blood and subsequently detect cancer related exosomes in a continuous manner. this consists of two parts. the first part is a channel for extracting plasma containing exosomes from whole blood, and the second part is a channel combined with an electrochemical sensor for multiple detection of various exosomes in the extracted plasma. previously, a multi-orifice flow fractionation (moff) channel that consists of a series of expansion and contraction structures has been developed in our group. in this channel, the blood cells are moved to sides of channels by hydrodynamic forces and then are eliminated to outlets. at this time, the plasma is moved to the electrochemical sensor part, the exosomes in the plasma are captured to the electrodes immobilized with the specific antibodies and are quantified the amount of cancer-related exosomes. results: using this chip, blood cells were eliminated from the whole blood with over 90% of separation efficiency at 225 µl/min flow rate and exosomes were collected continuously with high recovery (~70%). in order to quantify various types of exosomes, a labelfree electrochemical biosensor with electrochemical impedance spectroscopy (eis) was used for the continuous detection of exosomes. the limit of detection was 1x10^6 exosomes/ml. summary/conclusion: the developed device is an integrated device capable of separating exosomes from whole blood with high purity and quantitating exosomes through the electrochemical sensor in a continuous manner. , 1800528, 2019) . the development of highthroughput techniques capable of simultaneously monitoring physical and biochemical properties of evs would significantly simplify and accelerate the characterization process. in this context, microfluidic technology is emerging as an attractive platform. here, we present a microfluidic device based on the combination of diffusion sizing and multi-wavelength fluorescence detection to simultaneously provide information on ev size, concentration and composition. methods: the diffusion of evs in the microfluidic channel provides information on their size distribution, and four different staining protocols with high signalto-noise ratios track different ev native molecules. evs are separated from unbound fluorophores directly during the microfluidic analysis, therefore avoiding the need for sample pretreatments and allowing to operate the device as a single-step immunoassay. results: the microfluidic device coupled with complementary staining techniques allows to individually detect and size particle populations with different ev components such as lipids, primary amines and the ev marker cd63. we demonstrate that this approach can probe the abundance of ev-specific markers and impurities such as lipoproteins with high throughput and low sample consumption. summary/conclusion: we present a microfluidic technique capable of characterizing and quantifying evs at low costs, in a time-scale of minutes and requiring only up to 2 µl of non-pretreated sample. this method is an important complementary tool to the current array of biophysical methods for ev characterization, in particular for high-throughput screening applications. funding: h2020-eu.1.2.1-fet open programme via the grant agreement 801338. immunomagnetic isolation of specific subpopulations of exosomes for liquid biopsy via nano-architected porous materials introduction: exosomes offer the potential to reveal significant biological information in many areas of clinical importance by virtue of their rna contents and protein surface markers. this abstract reports the fabrication of a device for high throughput targeted immunomagnetic capture of exosomes via the use of highly-ordered nano-architected porous metal lattice materials. methods: we have invented a fabrication technique to precisely make millions of nanoscale exosome sorting devices that can operate on unprocessed plasma. each nanoscale device can precisely sort targeted exosomes from background vesicles but is too slow for practical use individually. however, the operation of millions of these devices in parallel preserves the precision of nanoscale sorting while also enabling high throughput and robust use on raw plasma samples. the metal lattice within which these devices are contained is assembled via metal electroplating onto a selfassembled polystyrene bead lattice with face-centred cubic (fcc) symmetry with 600 nanometre pores. the devices feature a conformally-coated layer of nickel-iron with gold passivation atop a base layer of nickel, resulting in a lattice of millions of nanoscale pores capable of magnetic sorting of exosomes tagged via surface-marker-based immunomagnetic labelling with magnetic nanoparticles. results: compared to our previous work on immunomagnetic exosome capture via commercial track-etched membranes (tempo), this device offers superior capture due to increased surface pore density (>25x) and three-dimensional pore density (>1000x) alongside lower required sample volume due to decreased noncapturing volume in the device. finite-element analysis simulations show that strong magnetophoretic traps emerge at the pore boundaries in this structure between higher-permeability metals such as nickeliron permalloy and the lower-permeability sample fluid in the device. preliminary experimental data shows that this device can isolate iron nanoparticles in solution with >100x enrichment from input and 49x capture efficacy versus tempo. summary/conclusion: current methods of exosome isolation such as ultracentrifugation and column chromatography all suffer from low throughput and limited yield. the application of inverse opal materials towards exosome capture offers the potential for isolation of specific exosome populations from very low clinical sample volumes or sparse biological signals. micropatterned growth surface topography affects extracellular vesicle production colin l. hisey a , james hearn b , yohanes nursalim a , vanessa chang a , cherie blenkiron a and lawrence w. chamley a a the university of auckland, auckland, new zealand; b university of auckland, grafton, new zealand introduction: extracellular vesicles are micro and nanoscale packages released by all cells and play an important role in cell-to-cell communication by shuttling biomolecules to nearby and distant cells. however, producing enough evs for many in vitro studies using conventional tissue culture techniques can be challenging, and despite the success of some bioreactors in increasing ev-production, it is still unknown how many independent culture conditions like growth surface topography can alter the production and content of evs. methods: standard 150 mm petri dishes were patterned with 2 µm tall polystyrene microtracks spaced by 5, 10 and 20 µm across a 100 mm area using standard microfabrication techniques including photolithography, soft lithography and microtransfer printing. the micropatterns were characterized with sem and profilometry, then activated with oxygen plasma and uv sterilized. mdamb231 cells were seeded onto patterned and smooth (control) dishes and grown in serum-free media for the final 48 hours of culture. evs were isolated using sequential ultracentrifugation of conditioned media and characterized using nta, tem and western blot. cell morphology was imaged using immunocytochemistry and single cell migration was characterized using time-lapse microscopy and manual single cell tracking in fiji. results: we demonstrate the simple and repeatable fabrication of microtracks across a large surface area in order to culture cells on topographically patterned growth surfaces. furthermore, we show that the 5 µm spacing produced significantly more evs than other patterns as well as the highest cell aspect ratio and average single cell migration speed (p < .05). summary/conclusion: these findings have implications in both biomanufacturing of evs and potentially in enhancing the biomimicry of evs produced in vitro. however, further experimentation to assess the differences in cargo on patterned growth surface topographies compared to conventional methods is still required. funding: this project was funded by the maurice and phyllis paykel trust. using miscroscale thermophoresis and surface plasmon resonance to measure the interactions of extracellular vesicles mst is a quick method, easy to handle, has a low sample consumption, has no limitation on molecule size, and enables measurements in solution, either in various buffers or complex biological liquids. these properties make mst an interesting tool for research of extracellular vesicles (evs); therefore, our aim is to apply this method to evs. methods: evs were isolated from jurkat cell line by differential centrifugation. microscale thermophoresis (mst) and surface plasmon resonance (spr) were used to analyse the interaction between antibody and evs. results: we have demonstrated that interactions of evs with antibodies could be analysed by mst. however, the tiny glass capillaries for sample mounting represent a challenge due to adhesion of evs to their surface. we have tested commercial capillaries as well as prepared capillaries in house coated by liposomes or bovine serum albumin. the interactions between evs and antibodies were confirmed by surface plasmon resonance (spr), which is an established method for studying the interactions of evs. introduction: the isolation of extracellular vesicles (evs) from cell culture supernatants and complex body fluids, such as blood and urine, is of high importance for ev research as well as for future medical applications in diagnostics and therapy. nevertheless, it is still challenging to reach the desired recovery, purity and specificity due to many manual and time intensive sample preparation steps. conventional centrifugation for ev isolation or sample preparation prior to affinity-based separation methods can damage evs and cells, leading to misinterpretation of results or inactive evs. alternative field flow fractionation methods employing acoustic fields are highly promising, but so far limited to laboratory usage, based on a complex (moulding) fabrication and/or hardly reproducible. here, we present an innovative surface acoustic wave (saw)-based acoustofluidic device for gentle sorting of cells and particles. methods: our device consists of interdigital transducers patterned on a piezoelectric substrate generating saw propagating on the substrate surface. upon interaction of saw with our on-chip structured, fluid-loaden microchannels, an acoustic pressure field is developed across the fluid wherein particles are suspended. this pressure field can be employed to simply manipulate cells and particles based on their intrinsic properties, such as size, density and compressibility in continuous flow. the device is manufactured using precise and low-cost microtechnological methods and is suitable for reproducible mass fabrication. results: we demonstrated the separation of blood components, i.e. the sorting of erythrocytes and thrombocytes. furthermore, we could also show results on thrombocyte activation indicating a gentle separation without damaging these shear-sensitive cells, as well as first results on plasma separation from whole blood samples and nanoparticle sorting. summary/conclusion: our unique acoustofluidic sorting technology for complex suspensions has the potential to overcome the need for time-effective, cheap and gentle separation of evs. funding: this work was supported by efre infrapro project "champ: chip-based acoustofluidic medtech platform". nanophotonic platform for cancer-associated exosomal microrna detection introduction: exosomes have an important role in intercellular communication at physiological and pathological processes. their cargo includes micrornas (mirs), single-stranded non-coding rnas, involved in alterations on recipient cells, such as development of tumourous phenotype and metastasis. more particularly, mir-21 excels due to its association with several cancers. determining exosomal mirs as cancer indicators demands selective and accurate methods, which are not currently available or entail high costs. colorimetric photonic-based assays are a promising label-free alternative, which dismisses complex apparatus for signal reading since biorecognition is detected by colour change. moreover, the clinical and economic systems have also been demanding a decrease on the green footprint of biosensors, requirement fulfilled with naturally derived biomaterials. methods: herein, the biosensor is constructed on a biopolymer matrix to meet the requirements of an eco-friendly disposable device, and it is based on a photonic structure obtained by imprinting a nanopattern on the polymer surface. then, the surface is functionalized with the complementary oligonucleotide sequence of mir-21 as sensing probe. a labelfree detection is thus envisioned and the sensor performance is evaluated by changes in the optical properties when the target is present. results: the combination of biological materials conducted to a biosensor support with great flexibility and low water permeability, allowing easy surface functionalization. the self-reporting ability of the photonicbased sensor enables high intensity colours detected by naked eye. summary/conclusion: the alliance with the high selectivity of oligonucleotide hybridization is expected to offer great exosomal mir-21 recognition ability and an optimistic perspective for utilization in clinical setups. funding: the authors acknowledge the financial support from the european commission/h2020, through mindgap/fet-open/ga829040 project. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = 31). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and 10 ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine 37 distinct uev surface markers of cd9+/cd63+/cd81+ vesicles in a multiplexed bead-based manner including respective controls. the accuri c6 (bd) was utilized for data acquisition. for further misev2018-compliant characterization, cd9+/cd63+/cd81+ uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd209, all other 36 surface markers could be identified. the most abundant markers were cd9 and cd63, which were detected in 93% of samples, followed by cd133/1 (84%), cd326 (81%), cd81 and cd24 (77%, each). cd3 (42%), cd9, cd45 (39%), cd49e (32%) and cd81 showed similar relative median fluorescent intensities (rmfi), while cd63 yielded significantly higher (p = 0.009) and all other markers significantly lower rmfi (p < 0.011). tem and f-nta revealed cup-shaped vesicles (137 ± 22 nm) with 8.8 ± 7.0 e + 10 particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg101, cd9, cd63 and cd81 without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a , esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = 40) aged 40-70 yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a 3colour panel, which included annexin v (for the majority of circulating evs), cd41 (for plateletderived evs) and cd105 (for endothelialderived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and 10-yr cvd risk detected by qrisk2. results: ev numbers, as determined by nta, were strongly associated with bmi (r = 0.602, p < 0.001), blood pressure (systolic bp: r = 0.359, p = 0.023; diastolic bp: r = 0.550, p < 0.001) and plasma triacylglycerol levels (r = 0.703, p < 0.001). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = 0.330, p = 0.038). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining 49.4% of the variance for total ev numbers. an additional 9.3% of the variance in total ev numbers was predicted by diastolic bp. ancova of the 10-yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with 10-yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd4 + t cells were isolated from secondary lymphoid organs of foxp3-rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th1, th2, and th17), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, 15-day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for 5 days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th1, th2, th17) showed either no change in proliferation (th1) or decrease in proliferation (th2, th17), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < 0.0001). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th1), il4 and il13 (th2), or il17a and il17 f (th17). in contrast, exposure of tregs to cdc-evs resulted in~1000-fold increase in expression of il10, a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il-10 in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il-10. this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t32hl116273 prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from 42 patients treated with prostanoids (study group; n = 42, 50 ± 16 years, 70% female) and 38 patients not treated with prostanoids (control group; n = 38, 55 ± 19 years, 65% female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; 0.5 mm), adenosine diphosphate (adp; 6.5 µm) and thrombin receptor-activating peptide (trap; 32 µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd61+ and cd62p+; pevs), leukocytes (cd45+, levs) and endothelial cells (cd146+, eevs) were measured in plateletdepleted plasma by flow cytometry (a60-micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = 0.01) and lower concentrations of pevs and levs (p ≤ 0.05). furthermore, thrombus formation was delayed (p ≤ 0.003) and thrombus size was decreased (p = 0.008) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd61+ pevs (p = 0.04). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ 0.05) and the concentrations of pevs (p ≤ 0.08). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ 0.04). summary/conclusion: patients with pah treated with prostanoids have increased risk of bleeding both due to impaired platelet aggregation, ev release and thrombus formation, compared to patients not treated with prostanoids. antiplatelet effect of prostanoids varies: whereas epoprostenol decreases the release of pevs, treprostinil and iloprost impair platelet aggregation. funding: ag is supported by the national science centre, research project preludium 2018/31/n/ nz7/02260. evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni 15924. nanoflow cytometry identifies an imbalance of epithelium-and neutrophil-derived extracellular vesicles in the airway environment of paediatric cystic fibrosis patients brian dobosh, vincent giacalone, milton brown, lucas silva, lokesh guglani and rabindra tirouvanziam emory university, atlanta, usa introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from 17 cf children (<6 years of age). for nanoflow cytometry, evs were stained with di-8-anepps, and with epcam, cd66b and cd115 (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = 0.003, spearman's rho = 0.689). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = 0.001, rho = 0.857). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv15a0), emory paediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: 142 individuals were enrolled into our study, subdivided into a training (volunteer n = 27, pneumonia n = 12, sepsis n = 28) and testing cohort (volunteer n = 20, pneumonia n = 18, sepsis n = 37). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq2) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative real-time pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed 29 significantly regulated mirnas in pneumonia compared to volunteers, and 25 mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by 12 mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: 0.982, pneumonia: 0.965, sepsis: 0.992). mir-193a-5p (log2fc = 1.86, padj = 1.49e-6) and mir-542-3p (log2fc = 1.67, padj = 3.29e-5) differentiated between pneumonia and volunteers and mir-1246 (log2fc = −2.41, padj = 1.78e-04) between pneumonia and sepsis. expression levels of mir-193a-5p and mir-1246 were related to disease severity. mir-542-3p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was 73.33%. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study,3healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd63 and cd81 were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and 9 mirnas (mir-103a-p, mir-191-5p, mir-320a, mir-200b-3p, mir-185-5p, mir-223-3p, mir-21-5p, mir-155-5p, let-7 g-5p), were evaluated by rt-qpcr. after the optimization of the methodology, 10 healthy adults subjects and 10 patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd63 and cd81. however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna-320a showed a significant increased (p = 0.02) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir-320a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf2-active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid 2-related factor 2 (nrf2) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf2 in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf2-active exosomes, mscs were incubated with potent nrf2 activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express >95% positive msc markers (cd44, cd73, cd90, and cd105) and <5% express negative markers (cd45, cd31, cd14, cd19, or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd81, cd9 and tsg101. nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of 168.0 ± 6.5 nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf2-active human mscs increase exosome secretion by 54%, compared to nrf2-baseline mscs (p < 0.05). both nrf2-baseline and nrf2-active exosome treatment significantly reduced closure time to 15.5 and 14 days respectively, compared to 29.8 days for vehicle-treated wounds (p < 0.05). this reduction eliminated the delay in closure time compared to wounds of c57/b6 mice. nrf2-active exosome treatment of db/db wounds reduced closure time by a further 2.6 days compared to untreated c57/b6 wounds. at day 10, exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd31+ vessels compared to vehicle-treated wounds. summary/conclusion: enhancing nrf2 function in mscs multiplies exosome yield. our results demonstrate exosome-based therapies hold tremendous promise and warrant further investigation for rapid translation. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist1 is a key mediator of tumour cell metastasis. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist1. methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc3-ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc3-ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc3-ml lines stably overexpressing or deficient in twist1. results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc3-ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc3-ml overexpressing twist1 showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist1 expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. introduction: exercise is associated with various health benefits, including the prevention and management of obesity and cardiometabolic risk factors. however, a strong heterogeneity in the adaptive response to exercise training exists. differential response to exercise training might be mediated by myokines (proteins, nucleic acids, metabolites) that can be released directly into the systemic circulation, or packaged within extracellular vesicles (evs). the objective of this study was to evaluate if changes in evs after acute aerobic exercise (ae) were associated with the responders phenotype following 6-week resistance exercise (re) training. methods: this is a secondary analysis of plasma samples from the exit trial (clinical trial #02204670). eleven sedentary obese youth (15.7 ± 0.5 years, bmi ≥ 95th percentile underwent an acute bout of ae (60% heart rate reserve, 45 min). blood was collected before [time (at) −15, 0 min], during [at15, 30, 45 min], and after [15 min (at60), 75 min (at120)] exercise. afterwards, youth participated in 6-week re programme, and were categorized into responders or non-responders (nr) based on changes in insulin sensitivity (above or below 50 percentile). primary outcome: evs were isolated using size exclusion chromatography (izon®) at baseline (at0), immediately after ae (at45) and after recovery (at120). ev protein concentration, size, and zeta potential were analysed in a single-blind fashion. results: responders had larger evs (~141.1 nm) as opposed to nrs (~97.3 nm) at at0 (p < 0.05) and this pattern was maintained at at45 and at120, though not significant (p = 0.1). nrs displayed differential ev size distribution (peaks at 100 nm or 300 nm), while ev distribution was highest at 250 nm in responders. no difference in average zeta potential or total ev protein yield was observed between groups. an increase in ev yield with exercise time and recovery was observed in both groups. summary/conclusion: our preliminary data suggest that ev size is significantly increased after an acute bout of ae in obese youth responders. further research to delineate the role of evs as predictors of exercise adaptation is warranted. funding: funded by dream and research manitoba. using dual-fluorescent reporter mice to track tissue-specific extracellular vesicles andrea estrada, gabriella hehn, zackary valenti, christopher allen, nicole kruh-garcia and dan s. lark colorado state university, fort collins, usa introduction: extracellular vesicles (evs) from tissues like skeletal muscle (skm) and adipose tissue (at) have been implicated in human disease but are understudied. skm is likely a major player in ev biology as it accounts for~35% of total body mass. tools to define cellular ev origin are needed because tissues like skm are comprised of a variety of cell types. here, we describe our ongoing efforts using the dual fluorescent mg/mt mouse as a tool to analyse skm-myocyte derived evs. methods: wild-type (wt) and mg/mt mice were used for these studies. mg/mt mouse cells express membrane-tagged red (mt) or green (mg) fluorescent protein in the absence or presence of cre, respectively. we made skm myocyte mg expressing mice using a mouse expressing cre on the human skeletal actin promoter. blood was collected via cardiac puncture and platelet-free plasma was obtained via centrifugation. plasma evs were isolated using exoquick, exoquick-tc or size exclusion chromatography. skm and at were dissected into~5mm chunks, placed in serum-free dmem and incubated for 24 hours. tissuederived evs were isolated using exoquick-tc. ev abundance was determined with a horiba viewsizer. individual evs were analysed with a cytek aurora spectral flow cytometer. settings were optimized using polystyrene beads and spectral unmixing was performed to allow detection of mg and mt. results: in wt mice, skm releases >100 times more evs than adipose tissue per unit of mass (p < 0.01 using paired student's t-test). since skm is also a major component of total body mass, these data further emphasize the importance of skm-derived evs. skmderived evs from wt mice were not fluorescent (<0.1% of events). evs from mg/mt mouse skm overwhelmingly expressed mg (>95% of events) with negligible (< 1%) expression of mt. at-derived evs robustly expressed mt but lacked mg. summary/conclusion: these data provide "proof-ofprinciple" that mg and mt are readily incorporated into evs secreted ex vivo. surprisingly however, plasma evs from mg/mt mice expressed very little mg (~3%) or mt (~4%). this observation was confirmed with three separate isolation techniques. we are currently exploring possibilities to explain this finding, including: 1) modification of evs post-secretion, 2) clearance of fluorescent evs by the liver or 3) that evs secreted from tissues remain predominantly in the interstitial space. funding: this work was supported by an innovative project award from the american heart association (18ipa34110052) to dsl. endothelial cd36 delivery of fa loaded extracellular vesicles is critical for thermogenesis. introduction: membrane cd36 facilitates tissue fatty acid (fa) uptake. we recently found that endothelial cell (ec) cd36 controls muscle and adipose tissue fa uptake, and influences the tissue's metabolic phenotype. the mechanism for cd36-facilitated fa uptake is unknown. here we examined the role of ec cd36 in thermogenesis and in fa delivery to brown fat tissue. methods: adult male mice were housed individually, restricted from food during acute (4 hr) cold exposure (4°c) with core temperature monitored every 30 minutes. after 4 hours, animals were sacrificed and samples collected for analysis. for cellular studies, human microvascular (lonza) or primary murine microvascular ec were used. for primary cells, crude cell pellets from lung homogenates were purified using mouse-cd31 magnetic beads (miltenyi). for microscopy studies, alkyne fa (cayman) was added to cells and to enable visualization of internalized fas, click chemistry (invitrogen) used to label alkyne-fa with alexa 568. for radioactive studies, primary lung ec were serum starved for 5 hrs and incubated overnight with 3h-oleic acid bound to fa-free bsa (2:1 ratio). media was collected, clarified by centrifugation to remove microvesicles and debris. small extracellular vesicles (sevs) were isolated from clarified media using total exosome isolation reagent (invitrogen) and counted for radioactivity. results: basal core body temperatures are similar in mice lacking ec cd36 (eccd36-/-) compared to controls (cd36fl/fl). however, during cold exposure at 4°c , eccd36-/-are unable to maintain body temperature (p < 0.001). plasma free fa are higher in cold exposed eccd36-/-indicating fa clearance by brown fat is impaired. mitochondrial function and expression of thermogenic and mitochondrial genes in brown fat from eccd36-/-and cd36fl/fl mice were similar. these data suggested that endothelial delivery of fas is necessary for thermogenic maintenance of body temperature. to examine fa handling by ecs we used alkyne fas to visualize the process. we found that fas are transferred by microvascular ec through caveolae-mediated transcytosis involving src signalling and cav-1 phosphorylation. the internalized cav-1 and cd36 positive vesicles containing fas are released as sevs. to determine the dependence of cd36 on this process, we treated primary microvascular ec with radiolabeled fa and found that sevs secreted by cd36-/-cells contain less labelled-fa (p = 0.05). summary/conclusion: endothelial delivery of fa is critical for thermogenesis. our working model for the mechanism of fa uptake by brown adipose tissue is the following: endothelial cells transfer the fa through caveolae-mediated transcytosis and secrete small extracellular vesicles (sevs) that help deliver fas to brown adipocytes. funding: this work is supported by nih grants dk111175 and dk056341. introduction: diet-induced obesity modifies intestinal permeability leading to bacteria infiltration and to a decrease in the number of immune cells protecting mucosa. as orange consumption is beneficial for human health and used in preventive medicine, we determined whether orange juice-derived nanovesicles (onv) might be recommended as nutritional strategies for the treatment of intestinal complications associated with obesity. methods: onv isolated from fresh orange juices were characterized by lipidomic, metabolomic, microscopy, nta and for their stability during digestion. intestinal barrier (ib = caco-2 cells+ht-29 cells differentiated with oleic acid) were treated with onv and co-cultured with adipocytes to monitor ib fat absorption and release. obesity was induced in mice fed for 12 weeks with a high-fat high-sucrose diet (hfhs mice vs standart chow diet mice). then half of the hfhs mice were gavaged with 150 micrograms/day for 4 weeks. results: onv did not modify high-fat high-sucrose diet-induced obesity and insulin resistance but reversed diet-induced gut modifications. six hours post-gavage, onv accumulated preferentially in jejunum involved in lipid absorption. in jejunum, and no other intestinal region, onv increased villi size, restored immune response and decreased barrier permeability in hfhsd mice. in addition, onv-treated mice had increased expressions of acat2, angptl4 and dgat1, but a decreased expression of fabp2, fatp4, mtp vs hfhsd animals, which indicated that fat absorption, tg synthesis and chylomicron release were strongly reduced. similarly to other plant-derived nanovesicles, these results were likely associated with onv lipid and metabolite compositions (strong enrichment in bioactive phospholipids: pe, pa, pc, pi and leucine) as onv did not resist to harsh digestive conditions in vitro and were poorly incorporated in enterocytes. as the effects of onv on the decrease in tg content and epithelial cell growth were also observed in vitro, gut microbiota unlikely participate to these effects. summary/conclusion: onv are important bioactive compounds of orange juice and for the first time we demonstrated that they can modulate lipid metabolism in the intestinal barrier associated with morphological changes. interestingly onv treatment targets mtp and angptl4 mrnas, 2 therapeutic intestinal targets to reduce plasma lipids and for attenuating inflammation in gastrointestinal diseases. therefore, onv might be used to reduce the development of dyslipidemia-associated diseases and to restore intestinal functions in obese patients. funding: olga triballat institut; benjamin delessert institut, inrae institut. association, structure, and function of fibronectin in extracellular vesicles from hepatocytes xinlei li, ruju chen, sherri kemper and david brigstock nationwide children's hospital, columbus, usa introduction: we have shown that extracellular vesicles from normal hepatocytes have anti-fibrogenic activity and that they preferentially bind to hepatic stellate cells (hscs, the principal fibrosis-causing cell in the liver) and hepatocytes. in this study, our goal was to determine the molecular nature of the ev components involved in cell binding. fibronectin (fn1) is a key component of extracellular matrix, functioning in processes including cell adhesion, differentiation, and wound healing. two types of fn1 are present in vertebrates, of which the soluble plasma fn1 is derived principally from hepatocytes, while cell-associated fn1 is produced by numerous cell types. here we describe a novel function of plasma fn1 in facilitating binding of hepatocyte evs to target cells. methods: differential ultracentrifugation was used to collect evs released by parental mouse aml12 hepatocytes, fn1 ko aml12 cells in which fn1 was ablated using crispr-cas9, primary human or mouse hepatocytes, or human hepg2 cells, or from human or mouse serum. evs were characterized by nanosight tracking analysis (nta), western blot, iodixanol gradient ultracentrifugation, and mass spectrometry. the binding efficiency of pkh26-labelled evs from parental (ev-hep) or fn1 ko (ev-hepfn1 ko) aml12 cells was analysed in hepatocytes or hscs. swiss webster mice were injected with ccl4 for five weeks to induce liver fibrosis, with some mice also receiving i. p. administration of ev-hep or ev-hepfn1 ko over the last two weeks, followed by determination of hepatic fibrogenic genes by qrt-pcr. results: ev-hep or ev-hepfn1 ko were 50-200 nm in diameter and positive for common ev markers (cd63, cd9, flotillin-1). mass spectrometry showed that fn1 was the most abundant protein in ev-hep and comprised principally the plasma form. the abundant presence of ev fn1 was verified by western blot and co-immunoprecipitation with anti-cd9 or antiflotillin-1. western blot showed that fn1 was also abundant in evs from primary human or mouse hepatocytes, hepg2 cells, and human or mouse serum. fn1 and ev-hep co-sedimented at a density of~1.15 g/ml. ev-hepfn1 ko yield and size-range were similar to those of ev-hep, suggesting that ev biogenesis is fn1-independent. as compared to ev-hep, the binding of ev-hepfn1 ko to target cells was highly reduced whereas ev binding was independent of fn1 expression by the target cells themselves. both ev-hepfn1 ko and ev-hep were anti-fibrogenic in vivo but only ev-hep attenuated collagen 1⍺1 expression in mouse hscs in vitro. summary/conclusion: fn1 is abundantly associated with hepatocyte evs and facilitates ev binding to target hepatocytes or hscs. additional studies are needed to clarify the functional role of fn1 in mediating ev-hep anti-fibrogenic actions in vitro or in vivo. elevated glucose increases soluble and aggregated forms of human islet amyloid polypeptide in islet-derived extracellular vesicles -implications in type 2 diabetes and islet transplantation introduction: type 2 diabetes (t2d) is characterized by reduced beta cell mass and function. islet amyloid, formed by aggregation of human islet amyloid polypeptide (hiapp), contributes to progressive beta cell loss in t2d. amyloid also forms in human islets during pre-transplant culture and following transplantation in patients with type 1 diabetes (t1d) which is associated with graft failure. the cellular mechanisms underlying islet amyloid formation are still unclear. in this study, we examined the potential role of islet-derived extracellular vesicles (ev) in the clearance of soluble and aggregated (pro)iapp species from beta cells and amyloid formation. methods: human islets isolated from cadaveric pancreatic donors (n = 4 donors) and wild-type or hiappexpressing (hiapp+) transgenic mouse islets (n = 4/ group) were cultured in normal (5.5 mm) or elevated (11.1 mm) glucose to form amyloid. ev (exosomes) were isolated from culture medium using classical centrifugation and ultracentrifugation. purified ev were analysed by nanoparticle tracking analysis. western blot analysis and double immunogold transmission electron microscopy were performed to verify the presence of ev markers as well as (pro)hiapp species and oligomers (aggregates). results: human islets formed amyloid during culture with elevated glucose which was associated with progressive beta cell apoptosis. (pro)iapp species were detectable in ev released from human islets cultured in normal and elevated glucose. the latter markedly increased (pro)iapp content in islet-derived ev. interestingly, hiapp aggregates (oligomers) were present in the majority of ev released from human islets cultured in elevated glucose but were not detectable in islets cultured with normal glucose. similarly, ev released from hiapp+ mouse islets which formed amyloid during culture had higher (pro)iapp content compared to wild-type islet-derived ev. moreover, hiapp oligomers were present in ev derived from hiapp+ islets but not wt islets. summary/conclusion: in summary, our data show that (pro)iapp species are present in islet-derived ev and that elevated glucose increases (pro)hiapp and its aggregates in ev released from islets. islet-derived ev may play a key role in the process of amyloid formation in t2d and human islet grafts. funding: university of manitoba research grants program (urgp). on. contraction, but not glycolysis, regulates the size of skeletal muscle evs secreted ex vivo. colorado state university, fort collins, usa introduction: skeletal muscle (skm) is a metabolically active tissue and accounts for~35% of total human body mass. acute exercise increases secretion of extracellular vesicles (evs), but the mechanisms responsible are unknown. muscle contraction increases the demand for atp which requires intercellular communication in order to adapt. we hypothesized that this "metabolic stress" during contraction increases skm ev secretion. methods: we tested our hypothesis using an ex vivo ev secretion assay. all studies were approved by the colorado state university institutional animal care and use committee. vastus medialis muscle (skm) from male c57bl/6 j mice (n = 6) or female mt/mg mice (n = 6) was cut into~5 mg pieces and added to 12 well plates (~50 mg/well) filled with 1 ml of serum-free dmem and placed in a cell culture incubator at 37 c for 24 hours. skm from male mice was treated with 2-deoxyglucose (2-dg) (0.1 nm -100 mm) to induce metabolic stress via inhibition of glycolysis. skm from female mice was treated with 10um of blebbistatin (bleb), a contraction inhibitor. after incubation, skm mass was measured and conditioned media was centrifuged (3,000 x g for 15 min) to remove cell debris. evs were isolated using exoquick-tc. nta was performed on isolated evs using a horiba viewsizer 3000. ev secretion was normalized to tissue mass and culture media volume then reported as ([particle]/ml/mg tissue). statistical comparisons for 2-dg experiments were made using a repeated measures 1-way anova. bleb experiments were analysed using a paired student's t-test. results: there was a trend towards greater ev abundance (p = 0.07) as a function of 2-dg treatment, but no effect on ev diameter (p = 0.37). bleb treatment did not alter ev abundance (p = 0.69), but significantly reduced ev mean diameter (p = 0.007; 11% decrease; dmso: 114.9 ± 5.1 vs. bleb: 103.9 ± 4.2). summary/conclusion: contrary to our hypothesis, inhibition of glycolysis with 2-dg did not stimulate skm ev secretion. however, bleb did appear to promote the release of small evs and/or inhibit secretion of larger evs. ongoing efforts are focused on testing other metabolic stressors and defining how blebbistatin promotes small ev secretion. funding: american heart association grant to dsl (ipa34110052). introduction: extracellular vesicles (evs, exosomes) are nanovesicles (30-200 nm) secreted from various types of cells. because of vesicular encapsulation of mirnas and enzymes, the evs play crucial roles in cell-to-cell communication by delivering these functional molecules to other cells [1] . on the other hand, the evs are highly expected as next generation therapeutic tools due to pharmaceutical advantages such as controlled immunogenicity, effective usage of cell-tocell communication routes, artificial modification and encapsulation of functional molecules. however, cellular targeting and uptake efficacy of the evs are insufficient to be utilized as therapeutic tools [2, 3] . in this study, we newly developed evs decorated with cellpenetrating sc18 or (sc18)2 peptides, which are derived from the c-terminal domain of the cationic antimicrobial protein, cap18, because the peptides can be efficiently internalized by breast cancer cells. [4, 5] . methods: all peptides were prepared by fmoc-solid phase synthesis. secreted evs from cd63-gfp stably expressing hela cells were isolated by ultracentrifugation. cellular uptake of evs was analysed using a flow cytometer and a confocal laser microscope. encapsulation of saponin in the ev was conducted by electroporation. results: sc18 peptide is known as one of cell-penetrating peptides, and branched structure of sc18 peptides, (sc18)2, further enhances the cellular uptake [5] . in this research, we examined the effects of the peptide modification on cellular ev uptake, and modification of the sc18 or (sc18)2 peptides on ev membranes was conducted via stearyl moiety. as our results, increased macropinocytotic cellular uptake by modification of the peptides was successfully attained. especially, the modification of (sc18)2 peptides showed higher cellular uptake and macropinocytosis induction efficacy than that of sc18 peptides. in addition, anticancer protein, saporin toxin-encapsulated evs modified with the (sc18)2 peptides significantly enhanced their biological activity with dependency of glycosaminoglycan expression on targeted cells. summary/conclusion: the cell-penetrating (sc18)2 peptide-modified evs shows high abilities to be effectively internalized by cells and are applicable for intracellular delivery of therapeutic molecules. this study is expected to contribute to development of intracellular delivery techniques based on evs. [ introduction: rna therapeutics possess high potential which is yet to be realised, largely due to difficulties involved in delivery to the cytoplasm of target cells. extracellular vesicles (evs) possess numerous features that may help overcome this hurdle and have emerged as a promising rna delivery vehicle candidate. despite extensive research into the engineering of evs for rna delivery, little is known about how their intrinsic rna delivery efficiency compares to current synthetic rna delivery systems. using a novel crispr/cas9 based rna transfer fluorescent stoplight reporter system, we here compared the delivery efficiency of evs to state-of-the-art dlin-mc3-dma lipid nanoparticles (lnps). methods: evs were isolated from mda-mb-231 cells expressing either a targeting or non-targeting control sgrna and applied to hek293 t stoplight+ reporter cells. lnps containing targeting sgrna were titrated onto hek293 t stoplight+ reporter cells to determine the minimum effective dose. lnp and ev particles were characterized using nanoparticle tracking analysis, dynamic light scattering and zeta potential analysis. sgrna copy number was determined using rt-qpcr. results: evs were 140 ± 7 nm in diameter as measured by dls and possessed a negative surface charge of −25.3 ± 3.3 mv. rt-qpcr and nta analysis indicated that sgrna ev loading was low, with only 1 in 3.8e05 ± 3.5e05 evs containing a single sgrna copy. nevertheless, evs containing targeting sgrna induced significant reporter activation while evs containing non-targeting sgrna did not. lnps were 51 ± 0.6 nm in diameter and possessed a neutral charge. these particles also induced significant reporter activation when loaded with targeting sgrna. when delivered via evs, only between 4 to 54 sgrna copies per cell were required to induce statistically significant reporter activation. in contrast, the minimal effective sgrna dose when delivered by lnps was considerably higher at approximately 9e03 copies per cell. summary/conclusion: mda-mb-231 evs deliver rna in a highly efficient manner and are functional at sgrna concentrations several orders of magnitude lower than those required for lnp mediated delivery. this underlines the potential of evs as rna delivery vehicles and highlights the need to study the mechanisms by which evs achieve their efficiency in order for improved development of rna therapeutics. the role of circulating extracellular vesicles in patients with chronic chagas disease introduction: chagas disease is a neglected tropical disease (ntd) caused by the flagellated protozoan trypanosoma cruzi. it is a major public health problem in latin america, and it is now expanding over the globe through immigration of infected individuals. eukaryotic cells release extracellular vesicles (evs) that circulate in body fluids and have an important roles in intercellular communication, both in physiological and pathological conditions. objectives. our study proposes to characterize and to compare the circulating evs isolated from plasma of the chronic chagas disease (ccd) patients with healthy individuals (controls). methods: peripheral blood was collected from patients and controls in the presence of edta and evs enriched from plasma by differential ultracentrifugation. the obtained evs were characterized and quantified by nanoparticle tracking analysis (nta) and added to human thp-1 cells. after 48 h, the cell supernatants were analysed by elisa for the presence of cytokines. results: lower amounts of evs were obtained from ccd patients in comparison with control individuals. however, the same amount of evs of ccd were more capable of inducing cytokines such as ifn-gamma and il-17 in relation to controls. summary/conclusion: although less evs are present in the blood of ccd, these evs induce high inflammatory reactions on macrophages suggesting a possible role of these evs in the establishment of chronic disease. funding: supported by fapesp, cnpq and capes. extracellular vesiclesa trojan horse for therapeutic agent delivery introduction: extracellular vesicles (evs) may prove to be one of the optimal payload carriers for therapeutic agents. while they travel through the extracellular space, the ev's lipid membrane layer shields their luminal cargo from deleterious external factors. when autologous evs are used to protect this therapeutic cargo, little immunogenic effects are expected compared to viral vectors and artificial structures, such as liposomes. their usage is potentially manifold, and they are ubiquitously present in all body tissues and fluids. the key is to develop a manageable ev loading agent for adoptive transfer therapies. methods: to exploit the unique properties of evs, highly positively charged proteins were used to load them with multiple biomolecules, such as a cas9 protein or dicer substrate dsrna as a functional payload and to improve their apparently inadequate natural ability to deposit cargo into the cytoplasm of recipient cells. results: highly positively charged proteins can associate with and/or diffuse through a phospholipid bilayer (thompson et al. 2012) . when these kinds of charged proteins are mixed with isolated evs in vitro, they are loaded into the evs. the positive charge of the protein has the advantage that it can associate with negatively charged agents, such as rna species, and aids the associated molecule to also incorporate into the ev. moreover, the positive charge of the protein helps with cargo delivery, and thus overcoming the bottleneck of the ev's cargo to escape the endosome post-uptake in a recipient cell. self-quenching fluorescent lipid dyes demonstrated that discharge of the highly positive ev cargo into the cytoplasm is concomitant with lipid mixing between the membrane of evs and the membrane of the recipient cell. when egfp-expressing microglia were exposed to evs loaded with a dicer substrate dsrna able to silence egfp via the positively charged protein, the uptake of dicer substrate dsrna was concomitant with a decrease in egfp expression in the microglia. a similar result was achieved when evs were loaded with cas9 protein conjugated to the highly positively charged protein. post-uptake of these cas9-loaded evs, microglia expressing anti-egfp sgrna (single guide rna) lead to decreased egfp expression. summary/conclusion: our ev delivery technology has the capability of delivering multiple biomolecules, such as protein and rna cargo and demonstrates postuptake of the ev functionality of the ev delivered cargo in the recipient cell. hybrid extracellular vesiclesbiomimetic tool for drug delivery to repair endothelial cell dysfunction introduction: traditional drug delivery systems (dds) are usually based on liposomes, micelles or dendrimers. unfortunately, many dds cause side effects including organ toxicity and/or unexpected immune response. in living organisms, extracellular vesicles (evs) are responsible for delivering biologically active molecules to distant cells. in vitro loading of therapeutic compounds into evs is still not effective and needs developing new strategies. for these reasons we aimed to design hybrid extracellular vesicles (hevs) with high loading capacity for dds. methods: for hev synthesis, we used human endothelial derived evs. using freeze/thawing method we fused them with liposomes composed of cholesterol and one of the three lipids: dopc, sphingomyelin or phosphatidylserine. to confirm membrane fusion, we applied a spectroscopy ruler -fret (förster resonance energy transfer) and cryotem imaging technique. we characterized hevs using nta (for size distribution evaluation), dls (zeta potential) and western blot (for detection of evs markers). we evaluated loading efficiency using calcein as a model drug. additionally, we performed cytotoxicity tests. results: in the cryotem imaging, pure and homogenous hev population with a diameter of 65 ± 15 nm was detected. additionally, we observed changes in zeta potential and in size distribution after fusion. fret measurements showed increased fusion efficiency with the increasing number of freeze/thawing cycles and dependence on a lipid-to-protein ratio in evs. additionally, hev had higher loading efficiency than liposomes and sole evs and that their internalization by endothelial cells did not cause a cytotoxic effect. summary/conclusion: based on cryo-tem and fret, we confirmed that our fusion method of hybrid evs is effective and can be applied as a delivery platform for dds to endothelial cells. response to a range of stressors. the functional activity of these evs in recipient cells may, in part, be driven by changes to their biological cargoes. however, the molecular details of the underlying ev biogenesis and loading processes, and how this may vary in different conditions, is poorly understood. methods: we first studied the effect of oxidative stress on the functional activity of evs in recipient cells using cell viability and mitochondrial membrane potential assays in drosophila s2r+ cells. we then carried out total rna sequencing of ev and cellular rna under three stress conditions and compared results to existing data in mouse cells. further to this we have used a bioinformatic pipeline to identify sequence motifs enriched in evs under stress. results: functional assays indicated changes to cell viability and mitochondrial membrane potential in recipient cells, which were donor cell-stress dependent. subsequent characterisation of rna showed an enrichment of ribosomal rna in evs relative to cells, but no significant changes to other biotypes. comparative analysis has also uncovered a set of genes enriched in evs under oxidative stress, and a further subset whose enrichment may be evolutionarily conserved in mouse. we also identified potential ev-loading motifs which may assist in rna loading specifically under stress. summary/conclusion: we have shown that evs derived from oxidatively stressed cells show dose-dependent differences in rna cargo and identified potential sequence motifs that may have a role in its loading. we are now validating the biological significance of these findings by combining different in vivo approaches in drosophila. this will enable us to gain insights into the basic mechanisms which govern ev loading in different contexts, and ultimately the molecular mechanisms underlying ev-mediated intercellular communication. ishai luz a , bibek bhatta a , kanaga sabapathy b and tomer cooks c a ben-gurion university of the negev, beer-sheva, israel; b national cancer centre, singapore, singapore, singapore; c ben-gurion university, beer sheva, israel introduction: mutations in tp53 are considered one of the most frequent genetic alterations in human cancer. besides the abrogation of the wild-type (wt) p53-mediated tumour suppression, a distinct set of missense mutations was reported to endow mutant p53 proteins with novel activities termed gain-of-function (gof). even though mutations in tp53 are typically thought to arise in the tumour cells rather than in the stroma, the non-cell-autonomous effects of these mutants over the tumour microenvironment are poorly understood. in the presented studies, focusing on colon cancer as well as on lung cancer microbiome, we investigated intercellular interactions mediated by exosomes and outer membrane vesicles (omvs) in the context of cancers harbouring mutant p53. methods: p results: in the colon, tumour cells harbouring mutp53 were found to exert a non-cell-autonomous effect over macrophages. when exposed to tumour cells harbouring mutp53, monocytes became polarized towards a distinguished subset of macrophages characterized by tams-related markers. the mutant p53 affected tam were characterized as tnf-αlow/il-10 high, over expressing cd-206 and cd163, with decreased phagocytic ability and increased invasion and matrix degradation potency. investigating the exosomal transfer from mutp53 tumour cells to macrophages, revealed a mutp53-specific mirs signature led by mir-1246 promoting the tam phenotype and creating an invasive front together with tumour cells. mir-1246 was also found to be the top mutp53-associated mir in a cohort of 57 human colorectal resected tumours. separately, in two lung cancer cohorts, we identified a signature of microbiome members associated with p53 mutations. acidovorax temperans, a gram negative bacterium, was found to be abundant in tumours of patients with mutant p53. we found a significant increase in tumour volume in animals inoculated with acidovorax temperans as compared to sham treated animals, and increased lung weight as a percent of total body weight. these preliminary data indicate that acidovorax temperans contributes to lung tumorigenesis in the presence of activated k-ras and mutant p53. omvs shed by acidovorax temperans promoted inflammatory signalling in lung carcinoma cells and elevated cd47 expression on tumour cells and sirpα levels on macrophages. summary/conclusion: altogether, these findings are consistent with a microenvironmental role for specific "hot-spot" gof p53 mutants tightening the interaction between the tumour cell and the immune compartment in colon cancer. in both colon and lung cancer, mutant p53 facilitates cellular interactions within the tumour microenvironmets mediated by vesicles. funding: intramural funding from the national cancer institute, national institutes of health. lori zacharoff and mohamed el-naggar university of southern california, los angeles, usa introduction: the metal respiring bacterium s. oneidensis creates outer membrane extensions and outer membrane vesicles that are sculpted by the novel bar domain protein bdpa. these vesicles and extensions incorporate mutliheme cytochromes involved in extracellular electron transfer to metals and electrodes. however, the physiological relevance of incorporating these cytochromes into the higher order 3d architecture of a vesicle or extension is unknown. given that bar domains serve as a protein sorting mechanism in eukaryotes, we investigated the pathway crosstalk between bdpa and outer membrane multiheme cytochromes as means to understand the physiological significance of membrane architecture. methods: o this end, vesicle morphology and content was measured using dry weights, dynamic light scattering, fluorescence microscopy and comparative proteomics from wild type s. oneidensis and deletion strains. results: cells lacking bdpa make large amorphous vesicles that are dense with protein. in contrast, a strain lacking outer membrane cytochromes recruits less total protein into smaller vesicles. proteomics to show that both bdpa and multiheme cytochromes are involved in recruiting other proteins to outer membrane vesicles and have a reciprocal relationship. summary/conclusion: in the absence of bdpa, protein crowding has to become the main driving force of vesiculation and bdpa is essential for efficient incorporation of cytochromes. however, multiheme cytochromes are not only vesicular cargo, but are also important for shaping and loading vesicles. both of these situations make it clear that vesicles play a role in increasing the respiratory surface area of s. oneidensis cells. moving forward, we hope to be able to control bdpa and cytochrome levels for selective recruitment of technologically relevant payloads. introduction: fascioliasis caused by fasciola hepatica represents a major economic loss and clinical burden in cattle farming worldwide. extracellular vesicles (evs) contain pathogen-derived molecules that represent novel biomarkers of disease. in the present study, we have identified potential new biomarkers of f. hepatica infection in evs present in sera of infected cattle. methods: parasites and sera were obtained from local abattoirs (valencia, spain, and medellin, colombia, respectively). 38 sera from infected and 32 from healthy animals. parasites were cultured, and evs obtained by sizeexclusion chromatography (sec) and characterized by nta, tem and proteomic profiling. recombinant proteins from f. hepatica evs (enolase and fh16.5 tegumentary protein) were produced, and coupled to magnetic beads. measurement of bovine igg antibodies was performed using luminex bead array technology. results: a total of 239 proteins were identified associated with evs as shown by the presence of typical ev-markers (tsg101, alix, cd63). two parasite proteins, enolase and the fh16.5 tegumentary protein were produced as recombinant proteins and used for detection of cattle igg employing luminex bead array technology. interestingly, significant differences were found in the fluorescence values of both recombinant proteins allowing discrimination between sera from infected and non-infected cattle. the use of the fh16.5 protein generated a highly significant difference between the two groups (p value = 0.003614); as did enolase (p value was 0.02294). summary/conclusion: this study demonstrates the usefulness of ev proteins as new biomarkers for early diagnosis of helminth infections using multiplex assays, a technology that may also be applied to other parasite ev molecules. life stage-specific glycosylation of schistosome-derived extracellular vesicles introduction: glycans play an essential role in pathogen-host interactions. larvae and adult worms from schistosoma mansoni release distinct subsets of glycoconjugates as excretory/secretory (es) products. extracellular vesicles (evs) are also among the es products. we recently found that schistosomuladerived evs are glycosylated and bind human dendritic cells via c-type lectin receptor (clr) dc-sign, leading to increased il-10 and il-12 release. here we investigated the glycosylation profile of evs released by s. mansoni adult worms, compared this to schistosomula evs, and addressed how this may affect parasite-host interactions via clrs. methods: evs from cultured s. mansoni parasites were obtained by ultracentrifugation and purified with iodixanol density gradients. isolated evs were analysed by nta and cryo em. n-glycan and lipid glycan content was determined by mass spectrometry. density gradient fractions with evs were loaded onto sds-page gels followed by western blot (wb) analysis using anti-glycan monoclonal antibodies (mabs). results: cryo em showed that adult worm evs lacked the long thin filaments that are characteristic for schistosomula evs. additionally, in contrast to schistosomula evs, glycolipids could not be detected in the adult worm evs. mass spectrometry analysis showed that the most abundant n-glycans in the adult worm evs contained galnacβ1-4glcnac (lacdinac, ldn) motifs, which correspond to previously published overall glycan profiles of this specific life stage. other differences in ev glycosylation between the two life stages were observed by wb using anti-glycan mabs: adult worm evs showed a paucimannosidic glycan motif whereas in the schistosomula evs galβ1-4(fucα1-3) glcnac (lewis x) was detected in line with previous ms analysis. introduction: phloem plays a central role in plant function, as it is the responsible for the translocation of photoassimilates from source-to sink-organs, and a long-distance route for signals distribution. due to the sap high nutrient content, sieve elements are primary target for plant pathogens and pests. in this work we aimed to isolate and characterize extracellular vesicles (evs) from cucumis melo phloem sap, derived from plant either exposed or not to the melon aphid, aphis gossypii (hemiptera: aphididae). methods: phloem exudates from 5-week-old melon plants, either uninfested or infested with adults of a. gossypii (n = 15, 4 replicates each), were collected by cutting the stem with a sterile razor blade between first and second expanded leaf from the top. evs were isolated by size exclusion chromatography, and analysed by nanoparticle tracking analysis (nta) and transmission electron microscopy. evs proteome was determined by quantitative mass spectrometry. results: evs from phloem sap were successfully isolated in every condition. no significant differences were detected among distinct samples, neither in particle concentration and size by nta, nor in protein concentration. most importantly, a total of 381 different proteins were identified in phloem sap evs, including 152 present in exosome databases (exocarta). on top of that, 44 differentially expressed proteins were identified in evs derived from aphid infested or uninfested plants (p value < 0.05). summary/conclusion: understanding how plants trigger their defences against pests and pathogens is important to develop new control measures. the characterization of several proteins in evs from the phloem sap provide valuable information on long distance signalling in plants. moreover, as plants lack an immune system comparable to animals, the different protein content in phloem sap evs after exposure to aphids could indicate their important role in delivering inducible defences against invading pests and pathogens. extracellular vesicles from nematode species heligmosomoides bakeri and trichuris muris contain distinct small rnas that could enable niche specificity in the host introduction: gastrointestinal nematodes are extremely prevalent parasites that infect most animals and 24% of human population. their success as parasites is attributed to their ability to secrete diverse molecules that modulate the host immune system. extracellular vesicles (evs) are one of the immune modulatory compounds they release that directly modulate host cells. our goal is to understand how the small rna (srna) cargo underpins ev function, using a comparative analysis of ev cargo from diverse nematode species. methods: we first compared how different ev isolation methodologies (ultracentrifuge (uc), size fractionation, sucrose gradient floatation) effect the small rnas detected in h. bakeri evs using different library preparation kits (cleantag, truseq), with or without polyphosphatase treatment. we then compared this to small rna libraries from t. muris evs using comparable methods, uc ev purification, with or without polyphosphatase treatment and using the cleantag library preparation kit. results: evs from both species contained mirnas, however the mirna gene familes in h. bakeri and t. muris evs are distinct. the mirna content detected in ev samples collected by different purification protocols is robust. the largest difference in detected mirnas was found when comparing different library preparation kits. although both h. bakeri evs and t. muris evs were dominated by srnas derived from intergenic or repetitive elements in the parasite genomes, only in h. bakeri evs were these secondary sirnas. summary/conclusion: h. bakeri and t. muris evs contain distinct small rna cargos, which may underpin their ability to colonise different host niches, and/or modulate the host immune system differently. t. muris evs do not contain secondary sirnas, in contrast to h. bakeri, however they are dominated by srnas derived from intergenic or repetitive regions. comparative analysis of helminth evs could help pinpoint the srnas involved in cross-species communication. please provide any keywords if applicable.: nematode, cross-species communication, small rna introduction: extracellular vesicles (evs) are secreted from various cells including cancer cells and known to contain protein and small rnas including mirna isoforms (isomirs). therefore we also focused on isomirs including other small non-coding rnas for biomarker discovery. although liquid biopsies using small rnas are promising biomarkers for early detection of cancer, current approaches to detecting and analysing mirnas in the blood are still inadequate. artificial intelligence (ai) data analysis may provide better algorisms for diagnosing cancer. methods: small rnas were isolated from serum or purified evs using a mirneasy mini kit (qiagen) and quantified by using the ion s5™ next-generation sequencing system. (thermo fisher scientific). evs were purified using total exosome isolation reagent (invitrogen™). ai data analysis was performed using jmp® genomics and datarobot enterprise ai platform. results: three small rnas, isomir of mir-21-5p, mir-23a-3p, and trf-lys (ttt) were significantly upregulated in breast cancer patients compared with the healthy cancer-free individual. the combination algorithm using these three small rnas allows for a more accurate diagnosis of the area under the curve (auc) 0.92. to test the possibilities that these small rnas are derived from cancer cells, we isolated evs from the serum and performed ngs analysis to profiled serum small rnas in evs. interestingly we found that two small rnas, mir-21-5p and mir-23a-3p, also high in breast cancer evs, indicating that these small rnas were expected to be derived from cancer cells. in oesophagus cancer, we also performed ngs analysis and identified twenty-four mir/isomirs candidates for diagnostic biomarkers. a multiple regression model selected mir-30a-5p and two isomirs (mir-574-3p and mir-205-5p) . the auc of the panel index was 0.95. we also performed ai data analysis and discovered the novel algorisms that can diagnose breast and oesophagus cancer more accurately. summary/conclusion: we demonstrated combinations of circulating non-coding rnas containing evs potentially useful for the detection of early-stage breast and oesophagus cancers. in addition, the datarobot enterprise ai platform enables us to the more accurate diagnosis of cancers at the early stage. identification of novel ev-associated mirnas as toxic biomarkers in mouse introduction: recent findings reveal that extracellular vesicles (evs), secreted from cells, are circulating in the blood. evs are classified into exosomes (40-120 nm), microvesicles (50-1,000 nm) and apoptotic bodies (500-2,000 nm). evs contain mrnas, micrornas, and dnas and have the ability to transfer them from cell to cell. recently, especially in humans, the diagnostic accuracy of tumour cell type-specific evs as biomarkers is more than 90%. in addition, micrornas contained in the evs are being identified as specific biomarkers in blood for chemical-induced inflammation and organ damage. therefore, micrornas contained in the evs released into the blood from tissues and organs in response to adverse events such as chemical substances and medicine are expected to be useful as novel biomarkers for toxicity assessment. in this study, we aimed to identify target organs by comprehensive analysis of ev rnas in the blood of mice after chemical exposure to establish a highly sensitive "next generation type" toxicity test for chemical substances and medicine using ev rna in blood as a biomarker. methods: all animal studies were conducted in accordance with the helsinki declaration and the guidelines approved by the animal care committee of the national institute of health sciences. c57bl/6 j male mice (12 weeks) were orally dosed with ccl4 (vehicle, 7, 70 mg/kg). serum were separated from blood after 2, 4, 8 and 24 hours after ccl4 administration. the serum was centrifuged at 10,000 x g to remove cellular debris and subsequently ultracentrifuged 100,000 x g. the pellet is resuspended in pbs and ultracentrifuged 100,000 x g again. the comprehensive small rna-seq of collected evs were performed according to the manufacture's protocols. results: we succeeded in isolating more than 10 novel small rnas, which could be used as novel highly sensitive biomarkers for hepatotoxicity due to carbon tetrachloride (ccl4: 7 mg/kg & 70 mg/ kg). well known hepatotoxicity biomarkers, mirna-122 and mirna-192 were upregulated more than 1000-fold in the administration of 70 mg/kg ccl4, but not responded in the administration of 7 mg/kg ccl4. summary/conclusion: these results suggest that mir-122 and mir-192 are mainly released from liver to blood directly only in the administration of 70 mg/kg ccl4, while novel more sensitive hepatotoxicity biomarkers which responded in the administration of both 7 mg/kg and 70 mg/kg ccl4 should be included in the ev. our novel biomarkers will accelerate a rapid evaluation of chemical substances and medicine in nonclinical safety evaluation. introduction: advancements in sequencing technologies have allowed analysis of the genomic landscape of cancer using circulating cell-free(cf) dna. however, cfdna does not originate only from tumour cells. we recently demonstrated that most of the dna circulating in plasma of cancer patients is associated with large evs (l-evs), and that l-ev-associated dna reflects genomic aberrations of the cells from which l-evs arise. since l-evs are specifically released by tumour cells, we explore their potential to report cancer-specific genomic alterations in patient plasma and compare it to cfdna. methods: differential ultracentrifugation, tunable resistive pulse sensing, qubit dsdna high sensitivity assay, capillary electrophoresis, whole exome sequencing (1500-2000x), targeted sequencing (qiaseqtm), flow cytometry. results: we show here that l-evs in the size range of >1 micrometre are present exclusively in plasma obtained from cancer patients and absent in plasma from healthy donors. in agreement with this finding, double-stranded(ds) dna is detected only in l-ev fractions of patient plasma and not in those obtained from healthy donor plasma using the same protocol. we also demonstrate that the fragments of dsdna associated with circulating l-ev are larger in comparison with cfdna (>10,000 bp versus~170 bp). a large-scale analysis of l-ev dna obtained from plasma of patients with metastatic castration-resistant prostate cancer (mcrpc) as well as with non-small cell lung cancer (nsclc) demonstrates that dna associated with circulating l-evs reports cancer-specific genomic alterations in both types of cancer. we further investigate if l-evassociated dna is intra-or extravesicular and demonstrate that it is present in both forms. we finally compare the purity of the tumour signal in intravesicular l-ev dna, total l-ev dna, and cfdna obtained from patient plasma. summary/conclusion: our results demonstrate that circulating l-evs contain high quality, large molecular weight dna that contains cancer-specific genomic alterations, supporting the use of l-evs as a source of tumour-derived dna in plasma. introduction: epidermal growth factor receptor (egfr) mutation driven lung adenocarcinoma (ac) represents a unique subgroup that lends itself to treatment with oral egfr tyrosine kinase inhibitors. current methods that are used to detect these mutations (e.g. l858 r or the resistance mutation t790 m) involve invasive tumour biopsies or blood circulating tumour dna (ctdna) and cell free dna (cfdna). the sensitivity of blood ctdna and cfdna is limited by the frequency of genomic alterations in the egfr gene; additionally, ctdna does not reflect changes in the egfr protein, against which novel therapies are in development. there remains a need to develop bloodbased biomarkers that can circumvent these disadvantages and replace the more standard, invasive tumour biopsies. we propose the study of exosomes for treatment monitoring as well as to identify egfr resistance related genomic and proteomic changes. methods: we enrolled 10 patients with metastatic lung ac: 8 with egfr mutations and 2 without (control). from the patients with egfr mutant lung ac, we processed 25 blood samples through the patients' treatment course, using ultracentrifugation to isolate exosomes. we then used both droplet digital pcr (ddpcr) to test exosomal rna (exorna) for the mutation of interest and western blots to test protein resulting from exon 19 deletion or l858 r mutations. results: from 6 patients with egfr exon 19 deletion mutations, we detected identical mutations in exorna from 15/19 samples. exorna based mutational load increased and mirrored clinical progression in 2 patients. three patients whose cancer remained stable demonstrated a decrease in their exorna. one patient had blood drawn only at 2 points and was therefore not plotted. exorna from 2 patients with l858 r and t790 m mutations demonstrated the corresponding mutations; however, exorna did not mirror their disease course. we also demonstrated mutant egfr protein presence in exosomes from 3 patients. finally, we tested cfdna for egfr mutations from four matched samples using ddpcr. we detected matched mutations in exosomes in all four, while cfdna mutations were only detected in 2/4 patients. summary/conclusion: in summary, we detected egfr mutations in 19/23 exosome samples isolated from metastatic lung ac. our results set the stage for optimization of exorna methods and inform future experiments relating to exosomal cargo in patients with egfr mutant lung ac. identification of plasma-derived, ev-based biomarkers for glioblastoma introduction: glioblastoma multiforme (gbm) is the most malignant and aggressive primary brain cancer in adults, with an incidence of 3.2 per 100,000 people. currently, diagnosis is only performed via histopathological investigation of a tissue sample from a gbm lesion, complemented with molecular diagnostics for identification of select biomarkers. mri is the standard of care for follow-up and monitoring of treatment response. therefore, development of a "liquid biopsy" to obtain disease-relevant information from patient's body fluids is highly desirable. methods: we present the results from a clinical study in which extracellular vesicle (ev)-derived mrnas and long non-coding rnas were profiled from the plasma of gbm patients and control individuals. we obtained plasma from 8 patients at the time of initial diagnosis, and 8 matched controls by sex and age. ev-associated rna was isolated from 1-2 ml plasma and rna-seq was performed using our proprietary pipeline. sequencing data was analysed for differential gene expression. results: we observed 95 mrnas as differentially abundant between gbm and control samples, with 82 mrnas enriched in gbm samples and 13 mrnas enriched in control samples (p < 0.05). correlation based on differentially abundant mrnas separated gbm and control samples into two unique populations. eight differentially expressed mrnas were previously identified as part of the mesenchymal gbm subtype. these data, while preliminary, provide a potential basis for the further development of a noninvasive gbm gene panel test. summary/conclusion: we have identified a novel rna signature for gbm from plasma derived evs, which differs from previously identified biomarkers isolated from tissue. further work will refine this signature to enable detection, characterization, and patient monitoring for gbm with minimally invasive techniques. introduction: sjogren's syndrome (ss) is a systemic autoimmune disease in which inflammation progressively damages the moisture producing glands of the afflicted. 4 million americans are estimated to be suffering from the disease, 90% of which are women with an average age of 40. overlapping symptoms with other health conditions and co-morbidities make ss particularly difficult to diagnose, with average time to diagnosis of 3 years. saliva exosomal rna profiling has been primarily focused on small rnas and has been limited thus far due to the large contribution of sequencing reads from the oral microbiome. a noninvasive saliva exosomal rna (exorna) based test capable of diagnosis would be highly desirable. methods: we began by first developing a novel long rna-seq workflow to selectively enrich and profile human exosomal mrnas and long non-coding rnas (lncrnas) from saliva. we then profiled salivary exorna obtained from 7 ss patients and 7 healthy matched controls. finally, we performed differential gene expression analysis to obtain an exorna signature for ss. results: rna-seq data analysis demonstrated highly efficient enrichment of human transcriptome, with over 80% of reads mapping to the transcriptome. further rna biotype analysis showed over 75% of transcriptome reads mapped to protein coding genes and lncrnas. we detected over 10,000 mrnas and approx.1000 lncrnas. differential expression analysis (dex) of ss vs. healthy control exorna identified 455 upregulated genes, including 415 mrnas and 37 lncrnas (p < 0.05). 120 genes were found to be downregulated in ss, including 114 mrnas and 6 lncrnas. gene ontology analysis of dex genes revealed enrichment of genes involved in various immune system related pathways. most importantly, principal component analysis (pca) resulted in clear separation of ss patients from healthy controls. summary/conclusion: our optimized rna-seq workflow enables saliva-based liquid biopsy for biomarker discovery. the gene signature identified in this ongoing study could potentially provide a non-invasive molecular means of diagnosing sjogren's syndrome. introduction: increasing embryo implantation rates has become one of the greatest challenges in assisted reproduction techniques. usually an endometrial biopsy is done to identify a receptive endometrium, which prevents embryo transfer in the same cycle, as it is detrimental for the implantation. the implantation is a complex process, which requires a synchrony between the development of the embryo and the endometrium, but also, an adequate embryo-endometrial cross talk. the presence of extracellular vesicles (evs) as mediators of this communication has been describe in the endometrial fluid. therefore, we hypothesize that the molecular analysis of the content of the evs and companion molecules from endometrial fluid could be a non-invasive method to recognize an implantative endometrium and consequently improve the implantation rates. methods: the objective is to define a simple, sensitive and reproducible non-invasive ev-based method that allow the quick identification of an implantative endometrium by means of mirna analysis. for the establishment of a robust methodology for analysing evs from endometrial fluid in clinical settings, where the sample is limited and no sophisticated equipment is available, five different methodologies were compared in triplicate. two of them consisted in the direct extraction of rna while in the other three, before the rna extraction an enrichment of evs was done. smallrnaseq was performed to determine the most efficient method. once the best method was selected, it was applied in a set of real samples with different implantation outcome. the content of mirnas (mainly associated with evs) of endometrial fluid samples from women in whom the implantation was successful (n = 15) and unsuccessful (n = 15) were analysed. results: our results show that the protocols with a previous enrichment step of evs obtained a higher mirna expression. the results obtained from the differential analysis of the set of samples with different implantation outcome are being analysed and it is expected that the results will be available by the time this communication is presented. summary/conclusion: this work demonstrates that it is possible to obtain and analyse evs and evs-associated mirnas from a small volume of endometrial fluid samples, which allows the use of ev-mirnas as a low-invasive biomarkers for the detection of an implantative endometrium. funding: jip is supported by a predoctoral grant from the basque government. small rna cargo of evs is affected by hormone treatment in prostate cancer introduction: small rnas are recently reported as a regulator for prostate cancer progression to castration-resistant disease. our previous work has shown that evs protein cargo is affected by male steroid hormone, dihydrotestosterone (dht). in this study, we assess the small rna cargo of evs in response to androgen manipulations. methods: androgen receptor-positive lncaps are grown in css medium to deplete the androgens. media were then replaced with vesicle-depleted css medium ± 10 nm dihydrotestosterone (dht) ± 10 µm enzalutamide (enz) for 48 h. evs were isolated using sequential ultracentrifugation (2000 g for 20 min, 10,000 g for 30 min, 100,000 g for 2 h), washed once in pbs. protein and rna were collected from both parent cells and conditioned medium to allow direct comparison between s-evs cargo and cells. small rna ngs libraries were prepared using the illumina's truseq small rna library prep kit and single-end sequenced at a read length of 50 nucleotides (nt). fastq library files were processed using a custom-designed pipeline. adapters were removed using the cutadapt tool, trimmed reads were mapped with high stringency against ribosomal sequences using bowtie2. snorna and trna fragments were identified using the flaimapper software. remaining reads were mapped against the human genome hg38 using bowtie2. results: we found that the presence or absence of androgens does not significantly change the amount of total rna in small evs (s-evs). however, hormone stimulation altered the small rna content of s-evs, in parallel with our previous published data on ev protein cargo. dht increased the abundance of snorna in cells, while a reduction of snornas was observed in the s-evs fraction. interestingly, dht induced the formation of cell filopodia that are not inhibited by androgen inhibitor enzalutamide. pathway analysis indicates the p53 mediated regulation driven by mirnas found in s-evs upon exposure to dht. the expression profile of snorna and trna fragments in dht treated cells resembles results from clinical prostate cancer specimens. summary/conclusion: our findings show that androgen manipulation alters both s-ev derived protein and rna cargo. changes in the s-ev rna profile due to treatment with androgens are not identical to small rna profiles in parental cells, indicating a specific sorting mechanism of s-ev small rna upon androgen manipulation. further, dht induces the formation of cell filopodia irrespective of enzalutamide, suggesting cargo selection of s-evs. we conclude that small rna ev cargo can be utilised to as prostate cancer biomarkers in androgen targeted treatments. introduction: cancer immunotherapy, such as pd-l1 blockade, is a method to eliminate cancer cells. ectopic expression of pd-l1, on the surface of tumour cells, has been associated with tumour persistence and as an important predictor of therapy response. a test that, specifically and accurately, detects pd-l1 is critically important in order to identify patients that would benefit from these treatments. emerging evidence has shown that extracellular vesicles (ev) can carry immune checkpoint molecules, such as pd-l1, and whose expression have been correlated with tumour immunity response. with a multitude of commercially available antibodies identifying appropriate clones and associated assay is important in order to standardize the diagnostic modality used. methods: pd-l1 expressing cancer cell lines were used to generate evs. pd-l1-myc vector was transfected to generate an overexpression system. exoview® sensors containing different anti-pd-l1 clones were generated. samples (cell derived and plasma) were incubated on chips to allow the antibody to bind the antigen on the ev. after incubation, chips were immunolabeled with fluorescently labelled antibodies against pdl-1 or ev associated markers. exoview r100 reader was used to enumerate the evs captured on the sensor surface and analyse the expression of pdl-1 on single vesicle through fluorescence imaging. immunoprecipitation and mass spectrometry (ip/ms) were employed as an orthogonal method to verify the specificity of the assay. results: to study the detection efficiency of the antibodies, engineered pd-l1-myc evs were used. under these circumstances, all the tested antibodies were able to capture evs. when testing endogenous pd-l1 positive evs from different cancer cell lines, only 28.8 and 73 10 clones consistently bound to evs. in addition, evs derived from plasma demonstrated to be positive for pd-l1, however, only clone 28.8 was able to immobilize these evs. the results suggested that clone 28.8 could be a potential pd-l1 antibody to detect pd-l1 positive evs originating from various sources. to confirm these results, and assure the specificity of the antibody targeted ip/ms was employed. summary/conclusion: in combination with the exoview platform, anti-pd-l1 antibodies can be screened and potentially used to generate a non-invasive ev-specific assay that could detect this protein in patients. differences in extracellular vesicle protein cargo is dependent on head and neck squamous cell carcinoma cell of origin university of michigan, ann arbour, usa introduction: head and neck squamous cell carcinoma (hnscc) is the sixth most common, eighth most fatal cancer worldwide and includes cancers of the oropharynx, larynx, hypopharynx, and oral cavity. in 2017, there were over 49,000 new cases and 9,700 deaths estimated in the usa alone. despite recent advances in treatment, including radiation, chemotherapy, surgery, concurrent chemoradiation, and immunotherapy, many tumours develop resistance and progress. patients develop metastases or tumours recur locally or regionally; the 5-year overall survival rate for hnscc is only 40-50%. factors that contribute to poor survival for patients with hnscc include late stage diagnosis, lack of reliable markers for early stage detection, high level of biologic heterogeneity, and local recurrence and distant metastases after treatment. methods: this study used 8 representative hpv-positive and hpv-negative hnscc cell lines, one hpvtransformed cell line. and two non-cancer oral keratinocyte cell lines. evs were isolated using differential ultracentrifugation and peg precipitation/ultracentrifugation. evs were characterized by tem, nta, and wes protein analysis for reported ev markers. ev and whole cell lysates were assessed by lc-ms/ms analysis using the tandem mass tag-10plex kit. cluster analysis was performed on the fold-change peptide spectrum matches (psm) for the evs from the hnscc lines compared to the evs from the normal keratinocyte line (noksi). protein was measured using a capillarybased electrophoresis instrument. results: cd9 and annexinv were detected in all of the ev lysates tested, while calnexin was detected in all of the whole cell lysates and none of the ev samples tested. selected proteins stat3, hla-a, tenascin, e-cadherin, β catenin, cytokeratin 19, epha2, and cd59, and hpv-related markers p16, p53, rb, cyclin d1, and egfr were tested using the wes platform. evs from hpv-positive cell lines showed higher protein levels compared to evs from hpv-negative cell lines in stat3, hla-a, and tenascin. only kert19 demonstrated lower protein levels in evs from hpvnegative cell lines. of the common hpv-associated hnscc markers: egfr, p53, rb, cyclin d1 and p16, only egfr was positive in any the evs tested. the remaining proteins queried, e-cadherin, β catenin, epha2 and cd59 showed varying protein levels in evs from both hpv positive and hpv-negative cell lines. summary/conclusion: our findings suggest that these proteins may be potential hnscc ev markers that may be 1) selectively included in ev cargo for export from the cell as a strategy for metastasis, tumour cell survival, or modification of tumour microenvironment, or 2) representative of originating cell composition, which may be developed for diagnostic or prognostic use in clinical liquid biopsy applications. validation of antibodies on western blot for extracellular vesicles from biological human samples and cancer cell conditioned media the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: one of the major challenges in extracellular vesicles (evs) research is to prove the particles that are isolated are true evs, rather than other co-isolated contaminants, like lipoproteins. isev recommends using multiple assays to characterize evs. this study aims to validate the positive and negative protein markers for extracellular vesicles from plasma, urine and prostate cancer cell conditioned media (ccm). methods: membrane and cytosolic fractions of mcf7 cells served as positive and negative controls for all antibodies validated. evs were isolated from plasma of healthy volunteers, urine of healthy volunteers and ccm of pc-3 cells using differential ultracentrifugation. eight protein markers were assessed: positive markers cd63, cd81, cd9, flotillin1 (flot1), alix and tumour susceptibility gene 101 (tsg101), negative marker calnexin (canx), and contaminant markers apo-a1 for plasma and thp for urine. tetraspanins are small transmembrane proteins expressed in evs. flot1 is membrane protein that forms microdomains in the plasma. alix and tsg101, an accessory protein of the endosomal sorting complex required for transport, are involved in the biogenesis of evs. they are positive markers for evs. canx is in the membrane of the endoplasmic reticulum. apolipoprotein-a1 (apo-a1) is the protein components of lipoproteins, therefore it is marker of contamination for plasma ev. tamm-horsfall protein (thp) is contamination marker for urine ev, because it is most abundant protein in human urine. results: all antibodies were validated in the correct positive and negative control, thus confirmed as usable and reliable antibodies for western blot. in plasma ev, cd63, cd9, cd81 and flot1 were positive and canx and apo-a1 were negative. in urine evs, cd9, cd63, flot-1, alix and tsg101 were positive and canx and thp were negative. in ccm evs, cd9, cd63, flot1, alix and tsg101 were positive and canx was negative. summary/conclusion: we confirmed a high degree of ev purity from 3 sample types: urine, plasma, and ccm. of particular importance, we confirmed that evs isolated from biologic patient samples, plasma and urine, had low contamination. future work will use these methods to confirm purity of ev samples prior to addition analysis, such as examining ev cargo and biologic significance. proteomic study of mesenchymal stem cells derived exosomes modified using mir. introduction: the project we are working on is to modify the immunogenic profile of human cmms from the umbilical cord stroma through its stable transfection with anti-mir-21-5p, and therefore of the exosomes that these cells generate, for use in free-cell therapy to treat inflammatory process. methods: evs released from a primary culture of human umbilical cord mesenchymal stem cells and from primary culture of human umbilical cord mesenchymal stem cells mir21-/-modified through stable lentiviral transfection were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (tem) and measured by nanoparticles tracking analysis (nta). protein extraction from evs was made using ripa buffer and after checking protein integrity the total ev proteins. we performed a shotgun proteomic study using a tmt (10-plex) label of the total mir21-/-exosomes protein comparing it with normal exosomes. after labelling the ltq-orbitrap platform of proteored was needed for fraction injections and data acquisition. proteome discoverer 2.2 (thermo) was used for protein processing and quantification. results: a total of 1.861 proteins were identified at least with a unique peptide and we have able to establish the proteomic profile of mir21-/-exosomes against normal exosomes. we found out several protein modulated by mir21 and related to inflammation. summary/conclusion: we have able to establish the proteomic profile of mir21-/-exosomes against normal exosomes focusing on proteins involving inflammation process. all those results seem indicate that exosomes could be modified, which could be used as an anti-inflammatory free-cell therapy. funding: proteored concept test project grant. a novel extracellular vesicle isolation method used to discover urine liver disease biomarkers introduction: hepatocellular carcinoma (hcc) is the 6th most common cancer worldwide and the 3rd most common cause of cancer death; additionally, its incidence is increasing. while outcomes for early hcc are superior to those for late stage disease, early detection of hcc remains a challenge. current guidelines have suboptimal sensitivity and specificity. in this pilot study, we hypothesize that urine extracellular vesicles (evs) may identify candidate biomarkers towards the development of an inexpensive, widely accessible screening assay for the early detection of hcc. methods: urine samples from 20 healthy subjects, 19 subjects with cirrhosis, and 19 subjects with cirrhosis plus hcc were collected and processed using ymatrix columns to isolate ev-associated protein and mirna. protein was analysed using a tandem mass tag method on a thermo scientific orbitrap fusion mass spectrometer with comet/paws and edger processing. mirna was analysed using a targeted firefly microarray from abcam. differential expression and predictive modelling for the presence of hcc and cirrhosis was performed to identify candidate mirna and protein biomarkers. results: for mirna, 39 samples were eligible for analysis after low expression filtering. we used pair-wise ratios of 34 cancer-associated mirnas by gradient boosting of decision trees to develop a predictive model for hcc. our best model had a sensitivity and specificity of 0.93 and 0.92 respectively using 6 mirnas to distinguish hcc from cirrhosis. all samples were eligible for protein analysis. based on differential expression and biologic relevance, we identified 10 protein candidate biomarkers. interestingly, we found liver-selective proteins and known hcc/cirrhosis plasma/tissue markers, demonstrating proof-of-concept for the method. summary/conclusion: urine extracellular vesicles contain liver-selective proteins and known liver disease serum biomarkers as well as novel mirna and protein biomarkers that are significantly up-regulated in disease samples. the described candidate biomolecules may be easily accessible biomarkers with which to develop a sensitive and specific universal screening diagnostic for the early detection of cirrhosis and hcc. introduction: the peptidergic g-protein coupled receptors (gpcrs) are cell-signalling transmembrane proteins, which in their native form comprise of seven segments embedded in the cell membrane. this structural advancement is believed to be maintained in extracellular vesicles (evs). in autoimmune diseases, the presence of autoantibodies towards gpcrs is not uncommon, and to detect plasma autoantibodies, evs carrying gpcr will be used as template in a novel microarray screening tool. methods: purified evs from hek293 cells were printed on different types of surfaces; polymer coated glass slides and hydrophilic and hydrophobic plastic 96 well plates. five different print buffers were tested in a multiplex assays. spots containing evs were stained with biotinylated antibodies (cd9, cd63, cd81, adrβ2, hsp70, epcam and flotilin-1) followed by binding of cy5-labelled streptavidin and visualized microarray scanner. results: the outcome of these experiments was promising, as some of the chosen printing buffers showed increased tendencies to bind evs. the ev presence was verified with a panel of markers known to be present on small evs. in addition, the ev content of the adrenergic beta-2 receptor (adrβ2-receptor), which is a gpcr of interest in autoimmune diseases, was verified in some of the experimental setups. summary/conclusion: the approach of using evs as template in a screening tool possesses the potential to easily screen for autoimmune illness markers in diagnostic purposes. using the microarray technology allows the screening to be multivariate, specific and highly sensitive. circadian variation of extracellular vesicles secreted in urine: analysis of time point collection and normalization strategy. introduction: urinary extracellular vesicles (uevs) are an ideal source of biomarkers for kidney and urogenital diseases. despite the great deal of interest generated by uevs, little is known about its collection time and normalization approach. the majority of the studies on uevs focus on spot urine collection based on the assumption that it accurately reflects the renal function, although time point of collection is not standardized. therefore the practice to collect spot urine does not allow for calculating and standardizing accurately the uev excretion rate which may vary during the day. in addition, no research has been carried out yet to show the quantitative and qualitative difference of uevs between spot urine and 24 h collections.the aim of this study is to compare uevs excreted in all single voids during a 24 hour collection period and compare it with 24 hour collection performed. methods: uevs were enriched by differential centrifugation and electron microscopy, western blot, nanoparticle tracking analysis, tuneable resistive pulse sensing and imaging flow cytometry were used to quantify uevs and associated markers variation during the 24 hour. creatinine, urine osmolality and particle concentration were used to normalize the assessed analytes. results: electron microscopy showed a heterogeneous population of evs and western blot confirmed the presence of ev markers (tsg101, alix and cd9). rna was extracted by a column-based method (mirna extraction kit qiagen) and cel-39 mirna was spiked in each sample. a multiparametric detection of nephron markers podocalyxin, aquaporin-2 and uevs pan tetraspanins (cd9 + dc63 + cd81) was performed utilizing imaging flow cytometry. whereas the uev composition did not change across the 24 hours analysis, the quantity of uevs and related markers fluctuated during the day depending on the hydration and excretion rate.the results of a 24 hour urine collection reflected the average results of all single voids over a 24 hr period. creatinine and particle count normalization failed to normalize "outliers". summary/conclusion: this study represents the very first report which compares single void urine versus 24 hour uev analysis. we concluded that the 24 hour collection is the preferred choice for a robust and rigorous assessment of uevs and its associated markers. porcine body fluids differ in small extracellular vesicle counts: comparison of blood plasma, seminal plasma and cerebrospinal fluid as vesicle sources for proteomic analyses helena kupcova skalnikova a , jakub cervenka b , jaromir novak a , karolina turnovcova c , bozena levinska a , jana juhasova a , stefan juhas a and petr vodicka a a institute of animal physiology and genetics, czech academy of sciences, libechov, czech republic; b institute of animal physiology and genetics cas, v. v. i. libechov, libechov, czech republic; c analysis tools were used to identify in silico biological pathways and functions governed by detected mirnas. expression of putative targets of selected mirnas was tested using qpcr after in vitro delivery of uterine evs to ptr cells. results: careful characterisation confirmed that uterine lumen is enriched with a diverse population of evs caring mirnas. interestingly, 36 out of 79 detected mirnas showed difference in abundance between tested days of pregnancy and half of them was exclusively detected on d16. identified mirnas were characterized as potent regulators of cellular development, growth, proliferation, and movement, in addition to their involvement in organismal and embryonic development. the expression of 20 genes identified as a possible mirna targets was tested after evs delivery to ptr cells in vitro. both down-(e.g., ptger4) and up-regulated (e.g., lifr) genes were found (p < 0.05); involved in the same molecular and cellular functions enriched by detected mirnas. methods: evs were harvested from wild type and arrdc4-/-epididymal cells using differential ultracentrifugation, then characterised using nanoparticle tracking analysis and transmission electron microscopy. sperm motility was measured using computer assisted sperm analysis and imagej. fertilisation capacity was measured using the following assays: capacitation-associated tyrosine phosphorylation, calcium ionophore induced acrosome reaction, zona pellucida binding assay and in vitro fertilization with time-lapse imaging of embryo development. immunohistochemistry was also used to visualise two pronuclei formation and blastocyst morphology. arrdc4-/-sperm was supplemented with wild type evs in the above assays to assess whether they could restore function. results: sperm from arrdc4-/-mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as evidenced by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and production of two-cell embryos. we observed a significant reduction in ev production by arrdc4-/-epididymal epithelial cells, and addition of wild type evs to arrdc4-/-sperm dampens the acrosome reaction and restores zona pellucida binding. introduction: gestational diabetes (gdm) is among the most common pregnancy complications. despite treatment, up to 25% of pregnancies complicated by gdm result in infants being born large-for-gestational-age (lga). this not only causes problems at birth but predisposes offspring to developing cardio-metabolic disease in adulthood. there are no treatments for lga as the cause is unclear, although it is associated with altered placental vascular development. micrornas (mirnas) regulate placental development; they are produced within cells but can be released into the circulation inside evs, which in turn can be transported into target cells and tissues to influence cellular processes. we aimed to characterise circulating evs in pregnancies complicated by gdm-lga and determine if ev-derived mirnas have the potential to influence placental development. methods: maternal serum and plasma samples were collected from women with pregnancies complicated by gdm at 24-32 weeks gestation; placental tissue was collected at delivery and birth outcomes recorded. serum and plasma evs were isolated and characterised by electron microscopy (shape), nanoparticle tracking analysis (nta; size/concentration), and western blotting (ev-enriched proteins). mirna qpcr arrays were performed on evs. mirnas were quantified in placental tissue via qpcr. results: em and western blotting confirmed isolation of evs and nta revealed no significant difference in size/ concentration in gdm-lga pregnancies (n = 7) compared to gdm-aga (n = 13; p > 0.05). several ev mirnas were altered in maternal circulation in gdm-lga compared to gdm-aga (n = 7/group; >twofoldchange; p < 0.05), including four skeletal muscle-specific "myomirs": mir-1-3p, mir-133a-3p, mir-133b, and mir-499a-3p (all increased). all four myomirs were present in placenta but only mir-1-3p was significantly altered in gdm-lga compared to gdm-aga (n = 12-14/group; p < 0.05). summary/conclusion: ev-bound myomirs could have predictive value for aberrant foetal growth in cases of gdm. mir-1-3p regulates vascular development in other systems, so we propose that mir-1-3p contributes to lga by influencing placental vascular development, however further work is required to establish this. introduction: seminal plasma is particularly rich in extra cellular vesicles. myelinosomes are membranous organelles described throughout the seminiferous epithelium of the testis but never reported in semen. the aim of this study was to look for the presence of myelinosome vesicles in human seminal plasma. methods: because of the viscosity of seminal gel and its water-holding capacity, classical transmission electron microscopy does not seem to be an optimal technique to reveal the presence of myelinosomes in this fluid. cryo-electron microscopy is a technique that allows visualization of nanosized structures without prior fixation or addition of heavy metals for contrast. the sample is therefore visualized as close to its native state as possible. using standard myelinosome preparation from tm4 sertoli cells, we first analysed the appearance of "standard" native myelinosomes by cryo em and then compared it with the vesicles from human seminal plasma samples. results: we have specified by cry-em the morphological aspect of "standard" myelinosomes isolated from the culture media of tm4 sertoli cells. the vesicles with the same morphological appearance were revealed in human seminal plasma specimens. summary/conclusion: myelinosomes are membranous organelles found in the seminiferous epithelium of the testis and secreted by the somatic sertoli cells in the lumen of the seminiferous tubules.the preparations from human seminal plasma contains a population of large ev (average diameter 200 nm) whose morphological appearance resemble those of myelinosomes. defining the specific biomarkers and functionalities of myelinosomes in human seminal plasma are the concerns to be addressed in our further research. introduction: more than one million patients worldwide suffer from tuberous sclerosis complex (tsc) and have mutations in either tsc1 or tsc2 genes. together, the tsc proteins regulate mtorc1 activity. all tsc patient post-mortem samples exhibit renal disease and 40% of patients with tsc experience a premature loss of renal function. mouse and human studies are incongruity with the second somatic hit mechanism of disease, because of the low percentage of cystic cells exhibiting loss of tsc expression. we posited that the loss of a tsc protein expression may alter extracellular vesicle (ev) biology and contribute to disease. methods: we used crispr/cas9 to disrupt the tsc2 gene in mouse inner medullary collecting duct (mimcd) cells, and isolated evs using gel filtration from the isogenic cell lines. we characterized the evs using tunable resistive pulse sensing (trps), dynamic light scattering (dls), transition electron microscopy (tem), and wester blot analysis. we further performed mass spectroscopy on the ev proteins. results: loss of the tsc2 gene in mimcd cells induced a greater than three-fold increase in ev production compared to the same cells having an intact tsc axis. electron microscopy confirmed the purity and spherical shape of evs. both trps and dls demonstrated that the isolated evs possessed a heterogenous size distribution. approximately 90% of the evs were in the 100-250 nm size range. western blot analysis using proteins isolated from the evs revealed the cellular proteins alix and tsg101, the transmembrane proteins cd63, cd81 and cd9, and the primary cilia-related hedgehog signalling-related proteins arl13b. proteomic analysis of evs identified a significant difference between the tsc2-intact and tsc2-deleted cells that correlated well with the increased production. summary/conclusion: evs may be involved in tissue homoeostasis and cause disease by overproduction and altered protein content. the evs released by renal cyst epithelia in tsc complex may serve as a tool to discover the mechanism of tsc cystogenesis and in developing potential therapeutic strategies. introduction: we have shown that evs derived from amniotic fluid stem cells (afsc) of mouse origin present therapeutic effect in an animal model of chronic kidney disease, alport syndrome (as). in light of clinical translation, we isolated afsc-evs of human origin, characterized their cargo and evaluated thier therapeutic effect in vivo. methods: human clonal afsc were derived from amniotic fluid collected after volunteer donors provided consent. evs were obtained from afsc and identity and purity were assessed by rna-seq and proteomics. potency of hafsc-evs was evaluated by performing in vivo studies. ev biodistribution was evaluated by mri and therapeutic effect by measuring renal function and mice life-span. bulk rna-seq was performed on glomeruli obtained from injected and non-injected mice to identify potential ev regulating targets. results: proteomic profiling identified 675 intact proteins and rna-seq data identified 2,535 mirs in hafsc-evs. hafsc-ev "fingerprint" was assessed by performing go analysis on the 100 most highly expressed proteins and mirs. the results identified pathways involved in tissue homoeostasis such as mtor pathway, tgfβ and vegf pathways. when injected in vivo into as mice, biodistribution studies showed that hafsc-evs localized in the kidney, corrected proteinuria. no side effects (including teratoma) were noted in the treated mice. rna-seq of glomeruli obtained from treated as mice showed similar gene expression patterns to wilt type mice, by cluster analysis. our data indicated that hevs highly modulated pathways involved in collagen and matrix deposition remodelling, in addition to downstream targets of vegf, fgf, tnf, angiotensin and preserved glomerular cells structure and function. summary/conclusion: our protocol for hevs derivation is reproducible and allows derivation of ev lots with the same identity (specific cargo of proteins and mirs) and potency (present therapeutic effect in as). hafsc-evs modulated signalling pathways that are central to maintaining glomerular homoeostasis and preserved glomeruli structure with improved kidney function. this suggests the possibility of using hafsc-evs as a new therapeutic option for treating renal failure in humans. introduction: recent studies have shown that stem cell-derived extracellular vesicles (msc-ev) therapy improves renal outcomes in models of acute ad chronic renal disease. however, to better investigate the molecular mechanisms of ev-induced regeneration, and to define new ev sources, devices that mimic 3d organ architecture and flow conditions are needed. the aim of our work is to evaluate the regenerative potential of naïve and engineered ev in a millifluidic in vitro 3d model of glomerular damage in continuous perfusion. methods: methods: we set a millifluidic in vitro 3d model of glomerular filtration, a three-layers structure composed by human podocytes and glomerular endothelial cells, and, in between, of a basement membrane of collagen type iv. the barrier thus formed is set up inside a bioreactor, in a closed milli-fluidic circuit in which fluid flows continuously at a certain flow rate. we reproduced different pathological conditions and tested the localization and effect of evs in a dynamic system. : results: we obtained a standardized protocol and an adequate configuration of the milli-fluidic circuit subject to continuous reperfusion. renal damage was induced by doxorubicin or by hypoxia-reperfusion injury. we evaluated uptake, cargo transfer and effect of naïve and mirna engineered msc-evs or of klotho engineered ineffective evs administered into the dynamic co-culture system. evs were able to pass through the system and to deliver to podocytes proregenerative factors, promoting survival and limiting permeability. introduction: worldwide, renal cell carcinoma (rcc) is 8th most common cancer in men and 10th most common in women. new biomarkers are needed to aid rcc-diagnosis, provide prognostic information, and to predict response to modern targeted therapies. extracellular vesicles (evs) are an emerging source of cancer biomarkers because all cells, including cancer cells, secrete evs into biofluids as blood and urine. however, benign cells contribute to ev populations isolated from blood and urine reducing the diseasespecificity. we have developed a protocol for ev isolation directly from human rcc tissue that can increase tumour-specificity of biomarkers. methods: we obtained technical and biological replicates from normal kidney tissue and clear cell rcc tissue. serum-free media was incubated with the specimens. a combination of differential centrifugation, filtration, and ultracentrifugation was used for ev isolation. evs were quantitated using two methods, allowing for comparison between nanosight ns300 and nanofcm. tem was used to determine presence of intact vesicles in the ev samples. presence of ev introduction: urothelial carcinoma (uc) is a malignant cancer that affects the urothelial cells, representing 90% of all bladder tumours. at diagnosis 75% of bladder cancers are non-muscle invasive tumours. importantly, upon transurethral resection of the bladder tumour, nearly 35-80% of these patients will experience disease relapse and 10-20% will progress to muscle invasive tumour, requiring thereby, a rigorous and expensive follow-up. currently, this is performed through the frequent use of highly invasive cystoscopy and the low sensitivity urine cytology. thus, innovative liquid biopsy-based biomarkers that circumvent these drawbacks are highly desirable for improved uc clinical management. here, we aim to implement a protocol for the isolation and characterization of extracellular vesicles (evs) from uc patients' urine samples. methods: a two-step protocol involving ultracentrifugation (uct) and by size-exclusion chromatography (sec) was optimized for urine samples. the isolated urine-derived evs from 9 uc patients were then characterized according to their size, concentration (nta), morphology (tem), protein amount (lowry method), presence of ev-associated and disease-associated protein markers (western blot). results: isolated urinary evs from uc patients had a size ranging from 50nm to 400 nm with characteristic ev morphology, express ev-associated markers as cd63 and hsp70 and were negative for cell debris markers. the recovery yield and purity of isolated evs following each isolation technique was characterized. upon uct, sec was required to deplete most of the ev-associated thp and albumin protein contaminants. some disease-associated protein markers were highly enriched in isolated urinary evs compared to crude urine. summary/conclusion: taken together, these results indicate that a two-step ev isolation protocol was properly implemented and validated in uc patients' urine samples. notably, several ev-associated disease biomarkers were detected in the urine of uc patients. this ev-based liquid biopsy might provide the means for real-time monitoring of residual disease and relapse in uc patients. introduction: glioblastoma multiforme (gbm) is a very aggressive type of brain tumour. different gbm molecular subtypes (proneural, mesenchymal and classical) often co-coexist within the same tumour, with the mesenchymal subtype driving the tumour progression. recently, our lab demonstrated that the cargo of extracellular vesicles (evs) could mirror the molecular background of the gbm cells from which they were derived. altogether, we believe that gbm cell-derived evs can be directly involved in the expansion of the mesenchymal signature in tumours, thus supporting gbm aggressiveness. methods: non-mesenchymal (t98 & u138) gbm cells were "primed" using evs derived from mesenchymallike (u87 & ln18) gbm cells. ev-primed gbm cells were then co-cultured with their non-primed counterparts to determine whether the mesenchymal signature can "spread" from cell to cell via evs. effect on cell proliferation, migration and invasion (in hyaluronic acid hydrogels) was assessed following ev treatment and co-culture. the expression of mesenchymal gbm markers was measured by western blotting. further mass spectrometry analysis of cell and ev content was undertaken to describe potential underlying mechanisms. results: co-culture with ev-primed gbm cells significantly increased proliferation and hydrogel invasiveness of non-mesenchymal cells. interestingly, the stimulating effect of co-culture was even stronger on the proliferation of ev-primed gbm cells. moreover, further proteomic analysis revealed that expression of mesenchymal gbm markers such as cd44 was increased in non-mesenchymal cells following coculture. summary/conclusion: our data suggest that evs from mesenchymal gbm cells can be uptaken by gbm cells from different subtypes, thus stimulating tumour progression. overall, we think the present study provides with new insights for the understanding of gbm recurrence and the development of potential therapeutic strategies. introduction: triple-negative breast cancer (tnbc) is the most aggressive form of breast cancer. previously we reported that the heterogenous population of evs released from tnbc cells promotes the growth and aggression of recipient cells. here we investigated if, by using compounds proposed to inhibit ev release i.e. calpeptin and y27632 (to block those budding at cell membrane) and gw4869 and manumycin a (to block evs from mvbs), we could reduce the associated transmission of aggressive phenotype. methods: evs were separated from medium conditioned by tnbc cell line hs578ts(i)8, using a discontinuous optiprep density gradient, after the cells were treatment for 48 hrs with the compounds listed above. evs (pooled fractions 3-9 with a density range of 1.03-1.16 g/ml) were characterised by nta, bca, lipid assay, immunoblot, tem and flow cytometry. to investigate the functional effects of the evs released, proliferation and migration assays were performed on hs578t and mda-mb-468 cells using the ev to cell ratios of 1 × 105 evs/3x103 cells, 1 × 106 evs/3x103 cells, 1 × 107 evs/3x103 cells to evaluate doseresponse. ev-track id ev190109 (score of 88%). results: gw4869 significantly (p = 0.035) decreased ev release from hs578 ts(i)8 cells. manumycin a and a combination of calpeptin and y27632 (combo) decreased ev release, but significance was not reached. conversely, calpeptin and y27632 actually increased ev release; but not significantly. of the reduced numbers of evs released following gw4869 treatment, hla-dr+ evs were significantly (p = 0.032) enriched. none of the evs analysed significantly changed hs578 t or mda-mb-468 growth rates. however, evs from cells treated with calpeptin (p = 0.007), gw4869 (p = 0.025), manumycin a (p = 0.01) and combo (p = 0.001) caused significant reduction in mda-mb-468 migration compared to the effects of evs from untreated cells. similarly, ev from cells treated with gw4869 (p = 0.011), and combo (p = 0.018) caused significant reduction in hs578 t migration. summary/conclusion: while gw4869 was the only compound that caused a significant decrease in quantities of ev released, the evs that continued to be released following treatment with gw4869 or calpeptin and y27632 significantly reduced migration of both recipient cell lines. funding: phd funding: 1252 tcd scholarship and carrick therapeutics ltd extracellular vesicles from highly metastatic lung cancer cells induce barrier impairment, permeability, and epithelial-to-mesenchymal plasticity in a 16-day mature bronchial epithelium purdue university, west lafayette, usa introduction: epithelial-to-mesenchymal (emt) transition plays an integral role in cancer metastasis, which is responsible for as much as 90% of cancer mortality. cancer exosomes induce emt in bronchial epithelial cells, however, the epithelial cells inhibit emt when allowed to form a mature epithelial barrier with apicalbasal polarity. it is not known if cancer-derived extracellular vesicles (evs) can induce emt and more importantly, barrier disruption in a mature epithelium. here, we show that evs from a highly metastatic lung cancer cell line (calu6) are) are not only sufficient to induce emt in non-tumorigenic bronchail epithelial cells (beas-2b), but are also capable of disrupting a 16-day mature bronchial epithelial barrier by significantly reducing teer, inducing sixfold increase in permeability and complete loss of e-cadherin at cellcell tight junctions. methods: beas-2b and calu6 evs were characterized using electron microscopy, nanosight and western blotting for exosome-specific features. for permeability studies, beas-2b cells were cultured in transwell for 16 days to establish an intact epitheliumconfirmed by measuring teer (trans-epithelial electrical resistance). intact beas-2b monolayers were treated with calu6 evs at 1, 10 and 20 μg/ml for 24 hrs, and barrier intactness and permeability were evaluated by measuring teer, apical-basolateral translocation of dextran beads and confocal imaging of tight junctions (e-cadherin). for emt experiments, beas-2b cells treated with calu6 evs at 1 and 10 μg/ml were evaluated for ecadherin and vimentin levels by qrt-pcr and western blot after 48 hrs. results: beas-2b and calu6 evs were enriched in 50-200 nm size range, and cd9 and cd81 were enriched in the ev fraction in contrast to the cell lysate and vice versa for gp96. calu6 evs significantly impaired 16day mature beas-2b monolayer's barrier properties, which at the highest dose caused 33% reduction in teer from 27.0 ± 2.5 to 18.2 ± 3.2 ω.cm2 (n = 4). this was further confirmed by~sixfold increase in dextran beads' apical-basolateral translocation in 30 min (14.8 ± 7 ng/ml in control vs 87.3 ± 51 ng/ml in treated) (n = 3) and complete loss of e-cadherin expression at cell-cell tight junctions (n = 3). at the transcript level, calu6 evs induced significant downregulation of e-cadherin by 36% and upregulation of vimentin (mesenchymal marker) twofold (n = 3) in beas-2b cells, indicating transition into mesenchymal phenotype. summary/conclusion: we demonstrated the involvement of evs derived from highly metastatic lung cancer cells in inducing emt in bronchial epithelial cells and epithelial barrier disruptionthe initial stage of the intravasation process. grp78 plays a crucial role in the extracellular vesicle-promoted radioresistance of irradiated head and neck cancer cells introduction: small evs released from irradiated head and neck squamous cell carcinoma (hnscc) cells increase resistance of recipient hnscc cells to radiation in vitro. we have identified the glucose-regulated protein 78 (grp78), a chaperone protein of the hsp70 family which is involved in cellular stress responses and associated with worse survival in head and neck cancer patients, as an essential component of the ev-mediated radioresistance. methods: small evs were isolated from conditioned medium from irradiated and non-irradiated bhy hnscc cells by combined microfiltration (0.22 µm) and differential ultracentrifugation. grp78 surface expression was measured by proteomic analysis, immunoblotting and bead-facs. radiation resistance of bhy cells was determined by a clonogenic survival assay. results: increased grp78 was identified on the surface of evs from irradiated cells. the increase in ev grp78 correlated with increased grp78 expression at the donor cell surface. the grp78 content of recipient cells also increased upon transfer of evs from irradiated, but not non-irradiated cells, ultimately leading to enhanced cell survival. to check a potential role of elevated grp78 in radiation resistance we overexpressed grp78. here the modest (3x) overexpression of grp78 was sufficient to confer an enhanced radioresistant phenotype to the bhy cells. a correlation between grp78-dependent increase of radioresistance and activation of the akt pathway is yet to be determined. summary/conclusion: our results suggest a pivotal role for ev-transferred grp78 in modulating the radiation response of recipient hnscc cells. radiation directly increases the cellular and vesicular grp78 levels, and subsequent ev-mediated transfer leads to enhanced grp78 levels and radioresistance in recipient cells. this study provides new mechanistic insights into the effects of evs in radiation response and elucidates an interesting target protein and novel strategies for the improvement of radiotherapy. 3d modelling of ev release in progressing prostate cancer introduction: the modelling of cancer progression should be capable to translate acquired knowledge of cell behaviour to the real human body conditions. however, the extracellular vesicles (evs) isolated from 2d cell models are commonly exploited in research. taking into account the specificity of the prostate cancer (pc) environment, and a strong need of early diagnosis of castrate-resistance by prostate cancer (crpc) patients, we suggest in-depth profiling of different ev subtypes isolated from 3d culture as a new tool to model the progressing pc. methods: cells from hormone-resistant prostate carcinoma 22-rv1 line were cultured in 2d and 3d conditions, using 3d coseedistm. acd plasma controlled for haemolysis and remaining platelets was taken from patients with pc and crpc. the 40 fractions of 4 ev subtypes from cell culture and plasma were obtained by differential centrifugation (dc) followed by iodixanol density gradient purification. each of the fractions was measured by nanoparticle tracking analysis (nta), tunable resistive pulse sensing (trps) followed by elisa. for that, cd63 and cd9 were used as ev markers, apob and apoa1 for lipoprotein contaminants control, and cd3, cd41 and psma as tissue-specific biomarkers for determination of fractions containing evs of different origin. ev-contained fractions were subjected to next generation sequencing (ngs). results: in 3d conditions, the 22-rv1 cells produce up to 1000-times higher ev number than in 2d. size and density distribution of evs derived from 3d cultures but not of 2d resembled plasma evs. size distribution and biomarker expression among different ev subtypes allowed distinguishing between pc and cprcderived samples, indicating a potential to translate these results into clinics for early cprc detection. summary/conclusion: this work demonstrates a new approach to study the secretome of a progressing pc under 3d conditions. the profiles of ev subtypes produced by cancer cells growing in a 3d spatial architecture resemble the profiles of plasma evs and can serve a useful tool for the establishment of new biomarkers. introduction: renal cell carcinoma (rcc) is the most common primary renal neoplasm, with over 80,000 cases in the us alone each year. early detection of rcc leads to consistently better patient outcomes, and extracellular vesicles (evs) isolated from patient samples may prove to be a valuable clinical tool in the future. evs are abundant in blood and urine and show a large amount of heterogeneity but are difficult to analyse due to their small size and difficulty in isolation. here, we employ a multiparametric analysis of ev surface markers to identify a set of markers that may prove clinically relevant in future studies. methods: rcc cell lines vok111, vok130, and vok151 were cultured in flasks containing 500 ml of ev-depleted media (10% fbs, centrifuged 18 hr x 100,000 g). when cells reached~75% confluency, the conditioned media was collected and spun at 2,500 g for 10 mins two times to deplete any remaining debris, leaving~450 ml of media. this media was concentrated to a final volume of~7 ml using a pall jumbosep 100 kda mwco filter. this concentrate was purified from protein by using an izon qev-10 column, collecting 5 ml fractions. protein content of each fraction was analysed using a280 absorbance while concentration and diameter distribution were determined through nanoparticle tracking analysis (nta). pooled samples made of the three most concentrated fractions were concentrated to a final volume of~190 µl using the pall microsep 100 kda filter and then used for analysis in the miltenyi macsplex exosome kit. flow cytometric data were generated by the cytoflex s and analysed using flowjo and mpapass software. these positive signals were verified through bead-only controls and titrations. results: the mpapass software allowed for heatmap generation, data reduction, clustering and visualization of expression patterns. of the 11 detection antibodies used across 39 capture beads, cd276, cd26, cd82, beta-2 microglobulin, and cd151 were found to be prevalent in these rcc evs. these markers were found to be co-expressed particularly with cd63, cd81, and cd29. summary/conclusion: the use of multiplex analysis allowed for detection of five distinctive surface markers found to be prevalent in evs collected from rcc cell lines. these results demonstrate the utility of multiplex analysis and mpapass software for identifying potential markers of interest and provide proteins that are worth exploring further. the next steps to this work will be developing custom multiplex arrays that tailor capture and detection of evs specifically for rcc pathology. low molecular weight protein tyrosine phosphatase (lmwptp) carried by colorectal cancer cells-derived extracellular vesicles as a player in tumour-educated human fibroblast university of campinas -unicamp, campinas, brazil introduction: extracellular vesicles (evs) are doublemembrane-bound nanovesicles released by cells playing a key role as mediators of intercellular communication. low molecular weight protein tyrosine phosphatase (lmwptp) is upregulated in several cancers type, including colorectal cancer (crc), and it has been correlated with aggressiveness, chemoresistance and poor prognostic. methods: the aim of this study was to determine whether crc cells release lmwptp-enriched-evs and influence tumour microenvironment-associated cells as a representative tumour education. crc cells, hct116 and ht29, were cultured in serum-free medium for 24 hours. conditioned medium was concentrated by ultrafiltration (mwco 10 kda) and evs were isolated by total exosome isolation reagent (invitrogen). evs were characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and western blotting (wb). lmwptp levels were analysed by wb and sandwich-elisa. to evaluate tumour education, hff-1 fibroblasts were used as recipient cells. the uptake of evs (pkh26 fluorescently labelled evs), proliferation (viability) and migration (wound healing assay) were analysed in a co-culture model of crc-derived evs and hff-1. results: nta showed a higher concentration of evs released by ht29. hct116 and ht29 evs displayed a mean diameter around 140 nm and a cup-shaped morphology. isolated evs were positive for evs-markers cd81 and tsg101 and negative for gm130 a non-evs marker. ht29 lineage as well as derived-evs are lmwptp-enriched in comparison to hct116 cells and evs. upon incubation, fluorescently hct116 and ht29 derived evs were internalized into hff-1 cells in a perinuclear region. evs derived from both cells increased the viability and proliferation of hff-1 cells. intriguingly, evs derived from ht29 promoted cell migration. summary/conclusion: in conclusion, for the first time, we showed that lmwptp can be carried by evs derived from crc cells and lmwptp-enriched-evs can modulate biological aspects of hff-1 fibroblast. overall, our findings point lmwptp out as important player in tumour-educated fibroblast. exosomal mir-181a inhibition by vincristine and prednisone in paediatric acute lymphoblastic leukaemia. introduction: vincristine and prednisone are standard agents in treatment of paediatric acute lymphocytic leukaemia (p-all). mechanistically, vincristine induces apoptosis by blocking microtubules formation, while prednisone binds to cytoplasmic receptors and inhibits dna synthesis, both of which lead to apoptosis. the effect of these agents on exosomal micro-rna expression and its functional regulation is not yet investigated. elevated levels of mir-181a in circulating exosomes (nanoparticles) has been shown to lead to progression in several cancers, including all. we have previously shown that leukaemia-derived exosomes induce leukaemia cell proliferation via up-regulating of mir-181a expression and silencing of exosomal mir-181a reverses this exosomeinduced cell proliferating effect. the objective is to investigate the effect of vincristine and prednisone on exosomal mi-r181a expression in all. methods: jm1, sup-b15, and nalm-6 leukaemic cell lines were treated in vitro with vincristine (0.1 to 4.0 µm) and prednisone (0.1 to 12.0 µm) in exo-free medium and apoptosis was measured by mts assay. total rna of exposed cell lines was isolated and cdna was prepared for mir-181a analysis. expression of mir-181a was analysed by q-pcr. exosomes from conditioned medium of exposed cell lines were isolated by ultracentrifugation method. purity and particle size of exosomes were confirmed by western blot and nanoparticle tracking analysis (nta) assay respectively. total exosomal rna was isolated from exosomes (exo-rna) by trizol method. synthesis of cdna was carried out with the miscript ii rt kit (qiagen). results: vincristine and prednisone promote apoptosis in leukaemia cell lines (jm1 and sup-b15) in a dosedependent manner. both cellular and exosomal mir-181a expression was down-regulated by vincristine and prednisone exposure in all three leukaemia cell lines (jm1, sup-b15, and nalm-6). these observations demonstrate that cellular mir-181a down regulation in the parental cells is stable and can be transferred to exosomes, confirming the concept that exosomes are the fingerprint of parent cells. summary/conclusion: our data suggest that the vincristine and prednisone anti-proliferative effect in p-all maybe induced by another yet unexplored pathway, that suppresses mir-181a at a cellular and exosomal level in p-all, resulting in apoptosis. funding: this project is supported by the dimartino family foundation. secreted extracellular vesicles from renal cell carcinoma cells anatoliy samoylenko, artem zhyvolozhnyi, eslam abdelrady, naveed ahmad, genevieve bart and seppo vainio oulu university, oulu, finland introduction: clear cell renal cell carcinoma (ccrcc) represents the most common form of kidney cancer and is among the most lethal of all genitourinary cancers. despite surgery and medication therapy, most patients with metastatic ccrcc have a poor prognosis. intratumoural hypoxia is a key factor involved in renal cancer progression and it is known to promote secretion of evs by many types of tumour cells. methods: rcc-derived renca cells, embryonic kidney derived ub cells, and primary mouse hepatocytes were used in the study. evs were purified from cell culture media by gradient ultracentrifugation, sequential ultracentrifugation and exo-spin™ columns. before ev isolation cells were kept for 24 h either under normoxia or hypoxia (1% oxygen). evs were analysed by transmission electron microscopy with negative staining and immunolabeling, by nanoparticle tracking analysis (nta) and western blotting. cells proliferation and viability were assayed by live cell imaging using incucyte zoom (essen bioscience), cell metabolic activity by seahorse xf analyser (agilent), rna expression by qpcr and ddpcr. proteins were identified by ultra-performance liquid chromatography-mass spectrometry (uplc-ms). rna libraries were made using nebnext small rna library prep kit, and sequenced on nextseq550 (illumina). results: we showed that hypoxia induced production of evs by rcc cells, and characterized differences in protein and rna content of evs generated by renca cells cultured under normoxic and hypoxic conditions. we also showed that rcc-produced vesicles modify key features of tumorigenesis (gene expression, metabolic activity, motility, and growth) of target cells. these data were obtained by using two target cell types: model mouse kidney cells and primary mouse hepatocytes, which represent typical site of rcc metastasis with an exceptionally poor prognosis. we proposed that a possible mechanism of ev action in rcc is related to changes in caveolin-1 function. we also tracked renca-derived evs in a chick embryo model and in a novel kidney organoid co-culture assay developed by our group (xu et al., 2017) . summary/conclusion: hypoxia may influence tumorigenic properties of rcc by changing rates of production and composition of evs. funding: the study was supported by finnish cancer foundation grants. exosomes synthesizing her2 mirna and engineered to adhere to her2 on tumour cells surface exhibit enhanced anti-tumour activity introduction: exosomes are small extracellular vesicles averaging 100-150 nm in diameter. they serve as a means of intercellular communication. typically they consist of structural proteins as well as selected proteins, mirnas, mrnas, and long noncoding rnas. thus in an earlier report this laboratory designed a mirna targeting a major herpes simplex virus regulatory protein. as predicted by the nucleotide packaging signal the mirnas were packed in exosomes and on exposure to infected cells significantly reduced virus yields. her2 (human epidermal growth factor receptor 2) plays an important role in the neoplasia of some breast cancers. the protein is exhibited on the cell surface and is the target of therapeutic antibodies. methods: firstly, we report on the construction of a mirna targeting the synthesis of her2 both in cells constitutively expressing her2 and in cells transfected with a plasmid encoding her2. secondly, we report that the mirna targeting the synthesis of her2 reduced the viability of her2 positive cancer cells both in cell culture and in implanted tumours. lastly, we enhanced the anti-tumour activity of the exosomes by binding to the exosome surface a ligand with affinity for the her2 on the surface of tumour cells. the 293-mir-her2 exosomes package with mirna designed to block her2 synthesis and deliver to cells. these exosomes kill cancer cells dependent on her2 for survival but have no effect on cells lacking her2 or which were engineered to have her2 but do not depend on it for survival. the 293-mir-xs-her2 exosomes carry in addition a peptide which enables the exosome to adhere her2 on the surface of the cancer cells. in consequence, these exosomes preferentially enter and kill cells exhibiting her2 on their surface. the exosomes with 293-mir-xs-her2 are significantly more effective in shrinking the size of her2-positive tumours implanted in mice than the 293-mir-her2 exosomes. summary/conclusion: our studies indicate that exosomes carrying mirna against her2 have no effect on her2 negative cells it was nevertheless desirable to increase the uptake of exosomes carrying the her2 mirnas by her2-positive tumour cells. to this end we modified the exosomes to exhibit on their surface a peptide that bound the exosomes to the her2 on the surface of cancer cells. in consequence, we significantly enhanced the uptake of exosomes carrying the mirnas directed against her2 by her2 positive cells. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd2015071414385495, shenzhen science and innovation commission project grants jcyj20170411 094933148, jcyj20180306173333907 to shenzhen international institute for biomedical research. systematic characterization of ovarian cancer-derived exosomes unveil mirnas interfering with cd8 + t cell activation introduction: cd8+ tumour-infiltrating lymphocytes (til) have been widely reported to correlate with cancer patient survival, including ovarian cancer. even with the presence of tils, immunotherapy has limited success in ovarian cancer. understanding the interaction between cd8+ til and tumour cells is thus important. our hypothesis is that tumour-derived exosomes are released and taken up by cd8+ til such that specific mirnas contained within modulate physiological processes that inhibit cd8 + t cell activation. we aim to identify mirnas carried in tumour-derived exosomes that inhibit cd8 + t cell activation in ovarian cancer. methods: we purified exosomes from nine ovarian cancer cell lines and stocked in high concentration. interferon-gamma (ifn-gamma expression screening was performed after 3 days of co-incubation of tumour derived exosomes, cd8 + t cells, and activators in conditioned medium. cell counts and viability were tested by trypan blue staining at day 0 and day 3. rna-seq for exosomes were generated to identify mirnas critical in differentiation effects on cd8 + t cell activations. microrna target matching uncovered target mrnas while enriched pathway analysis predicted potential signalling pathways involved. results: our ifn-gamma screening results indicated the exosomes exhibit different behaviours in interfering cd8 + t cell activation owing to different donors. exosomes derived from peo.1 and ovca432 cells have consistent polarized results in ifn-gamma expression. exosomes derived from peo.1 remained a low ifn-gamma expression and from ovca432 stayed at relatively high level. small rnas profiling analysis between the two cell lines identified 56 mirnas (p < 0.05), and 13 mirnas have been reported with validated targeting information, and 10 out of 13 have targets involved in immune signalling. 210 mrna targets were uncovered by target matching. cmap search identified complex connections among mrnas with the top 20 enriched pathways actively involved in cell cycle and immune related behaviours. summary/conclusion: our ifn-gamma screening identified crucial mirnas in ovarian cancer exosomes interfering cd8 + t cell activation. computational modelling on both experimental and public multiomics datasets predicted promising signalling pathways of tumour-immune crosstalk for functional validation. irradiation of breast cancer cells alters the quality of dna cargo in the exosomes that they produce sheila spada, paul zumbo, doron betel, tuo zhang, nils-petter rudqvist and sandra demaria weill cornell medicine, new york, usa introduction: irradiation of breast cancer cells with an immunogenic dose (8gyx3) leads to accumulation of cytosolic dna that is sensed by cgas leading to interferon type i (ifn-i) signalling via cgas/sting pathway [1] [2] [3] . we previously showed that tumour-derived exosomes (tex) secreted by irradiated (8gyx3) (rt-tex) but not untreated (ut-tex) tsa carcinoma cells carry dna that stimulates the production of ifn-i in recipient dendritic cells (dc) via the cgas/ sting pathway [4] . moreover, mice vaccination using rt-tex, but not ut-tex, elicited anti-tumour immune response inhibiting tumour growth [4] . here, we hypothesized that the differential ability of rt-tex and ut-tex to activate ifn-i in recipient dcs is due to qualitative differences in dna cargo of rt-tex compare to ut-tex. methods: the length of dna purified from tex and from the cytosolic fraction of tsa cells was measured by agilent bioanalyzer. the dna cargo of tex was analysed by whole-genome sequencing (wgs) and whole-genome bisulphite sequencing. the percentage of methylation of total dna in tsa cells was quantified by 5-methyl cytosine dna elisa kit. results: dna fragments with size between 60 and 250 bp were enriched in rt-tex compared to ut-tex, as well as in the cytosolic fraction of irradiated compared to mock-treated tsa cells. wgs revealed that the entire genome was represented in tex dna cargo, regardless of rt. more than 99% of tex dna was of nuclear origin, but mitochondrial dna was increased in rt-tex. interestingly, we found that rt decreases the level of methylation in both exosomal and total dna in tsa cells compared to the controls. summary/conclusion: these data support the hypothesis that immunogenic rt alters some characteristics of the exosomal dna cargo, mirroring molecular changes occurring in parent irradiated breast cancer cells. the enrichment in dna fragments of 60-250 bp in rt-tex is intriguing considering that cgas is optimally activated by dna in this length range [5] . we are currently investigating which features of the cargo dna that differ between ut-tex and rt-tex may explain the differential ability to induce ifn-i pathway activation in recipient dcs. the identification of a dna signature associated with the ability of tex to activate the cgas/sting pathway could provide a circulating biomarker of the rt-driven immunogenic tumour response. introduction: triple negative breast cancer (tnbc) is among the most difficult cancer subtypes to treat and continues to cause a high number of cancer-related deaths annually. extracellular vesicles (evs) transfer cell type-specific cargo and have important implications in disease initiation, therapy and outcome. upon treatment of cancer cells with low-dose chemotherapy, released evs are able to transfer phenotypic traits to other cancer cells. new treatment strategies for tnbc, like inhibitors of the er stress pathway (ire1) might impact on ev biogenesis, cargo delivery and response of cells in the cancer microenvironment. our aim is to identify immune modulatory alterations in breast cancer cells and cancer derived evs upon treatment with inhibitors of the er stress pathway. methods: human tnbc cell lines were treated with ire1 inhibitor mkc8866 and cells were analysed for immune modulatory surface markers, like hla-i, b7-h molecules and different integrins. mitochondrial and lysosomal activities were investigated by the use of a mito-and lysotracker and analysed by imagestream (isx) technology. extracellular vesicles were isolated from cell culture supernatants by sequential centrifugation, quantified by nanoparticle tracking (nta) and characterized by exosome bead array. single ev analysis of total cell free supernatants and of isolated evs was performed by isx and marker positive evs were quantified for absolute fluorescence signals and total amount by objectives/ml. ev uptake into t cells was investigated by the use of different ev labelling strategies. results: several immune relevant surface markers (hla-i and cd54) are downmodulated by ire1 inhibition across different cell lines. cell surface expressed cd63 and b7-h3 show cell line specific downmodulation profiles upon ire1 inhibitor treatment. other immunomodulatory marker such as b7-h1 and b7-h4, integrin cd29, cell adhesion-promoting cd146 and stemness/metastasis marker (cd44 and ssea) are unaltered on ire1 treated breast cancer cells. cancer cell derived evs were tetraspanin positive (cd9, cd63, cd81), similar in number and showed differential expression of immune markers upon ire1 treatment. mitochondrial and lysosomal activities were unaltered under ire1 inhibition, whereas cell proliferation was diminished. no breast cancer-derived ev uptake of externally labelled evs into healthy t cells could be detected. summary/conclusion: ongoing analyses focus on the multicolour analysis of multiple markers on single evs by imaging flow cytometry and on the functional impact of cancer derived evs on t cells delivered by ev receptor binding. funding: dagmar quandt is supported by the sfi (cúram research centre, 13/rc/2073), the european regional development fund and the dr. werner jackstädt-stiftung. chair: uta erdbrügger -university of virginia chair: larry harshyne -thomas jefferson university comparison of three isolation protocols to search extracellular vesicles signature in sickle cell disease patients introduction: sickle cell disease (scd) is an inherited disorder characterized by chronic haemolysis and continuous activation of different cell types. extracellular vesicles (evs) were described to be at increased levels in scd patient's plasma compared to healthy subjects and were associated with several clinical manifestations such as leg ulcers and stroke. scd patient's plasma has increased concentrations of haem, free-hb and other proteins and lipoproteins as chronic haemolysis consequence. here, we report the comparison of three mostly used isolation protocols to search ev signature in scd patient's plasma by flow cytometry. methods: blood samples were obtained from scd patients (n = 3) following wisgrill et al., (2016) protocol. three different ev isolation protocols were used: differential centrifugation (dc), ultracentrifugation (uc) and size-exclusion chromatography (sec). lactadherin and calcein-am were used to detect phosphatidylserine (ps)+ vesicles and membrane integrity, respectively. platelet-derived evs (pevs), endothelialderived evs (eevs), leucocyte-derived evs (levs) and monocyte-derived evs (mevs) were quantified. silica beads were used to define evs gate and samples were acquired in the cytoflex cytometer platform. results: the quantification of pevs in uc, dc and sec samples was, respectively, 31x106, 8,5x106 and 9,7x106 events/ml mean, eevs was 6,4x106, 1 × 106 and 4,3x106 events/ml mean, levs was 2x106, 6 × 105 and 1,3x106events/ml mean and mevs 5,7x105, 6,5x105 and 3,7x105 events/ml mean. uc samples demonstrated a higher concentration of evs, which could be more useful to functional studies than dc and sec, however, it took more time to separate than dc. dc was the fastest method to separate evs from plasma, being useful to study large patients cohorts, but showed the smallest overall number of evs. sec also demonstrated high capability to detect evs in plasma and the possibility of obtaining a purer sample, although it is the most expensive and time-consuming method among all tested. all evs populations were detected in the three protocols tested. summary/conclusion: in summary, all protocols tested were efficiently to detect evs in scd patient's plasma and the definition of the best protocol may vary based on the research aim and time and budget available. funding: fapesp 2014/00984-3. gabrielle lapping-carr, joanna gemel, yifan mao and eric beyer university of chicago, chicago, usa introduction: aberrant cell-cell interactions involving the endothelium are central to the pathophysiology of sickle cell disease (scd), including acute chest syndrome (acs), a deadly and unpredictable complication. we previously demonstrated that the plasma of scd patients contains increased circulating small extracellular vesicles (evs) compared to controls and that those vesicles can disrupt endothelial integrity in vitro by affecting adherens junctions and ve-cadherin. the current study was designed to examine the effects of those evs on other cellular junctions including tight (zonula occludens 1, zo-1) and gap junctions (con-nexin43, cx43) and to test the hypothesis that the junctions would be more severely affected by evs isolated from patients during an episode of acs than by ones isolated from the same patient at baseline. methods: we identified subjects with scd in our biobank who had plasma isolated at baseline and at the beginning of an admission for acs. evs were isolated from platelet free plasma using established methodologies. to determine the effects on endothelium, cultures of human microvascular endothelial cells were treated with evs for 48 h and studied by immunofluorescence, immunoblotting and rt-qpcr. gap junction-mediated intercellular communication was assessed following microinjection of lucifer yellow and neurobiotin. results: the distribution and abundance of zo-1 at the plasma membrane were minimally affected by scd evs. while baseline evs did not affect the distribution of cx43, evs isolated during an episode of acs caused loss of cx43 from the plasma membrane. the integrated intensity of cx43 membrane staining was decreased bỹ 20% following treatment with acs evs. cx43 protein decreased on average by 32%, cx43 mrna levels by 21% and neurobiotin transfer by 67-94% in cells treated with acs evs, compared to baseline evs. summary/conclusion: circulating evs in scd affect multiple components of endothelial junctions. gap junctions composed of cx43 are the most sensitive of the cellcell junctions, since their abundance and function are reduced by acs evs even when the endothelial monolayer appears intact. cx43-mediated intercellular communication may be an early and sensitive event in the endothelial disturbance caused by evs in scd patients. funding: nih ul1 tr000430, comer hospital rbc race funds, ted mullin fund. the effects of platelet concentrate storage time on extracellular vesicle interactions associated with fibrin clot formation in-vitro jamie nash a , christine saunders b , amanda davies a and philip james a a cardiff metropolitan university, cardiff, uk; b welsh blood service, velindre university nhs trust, cardiff, uk introduction: platelet concentrates (pcs) have been utilised for decades to prevent bleeding in thrombocytopenic patients and to stop active bleeding. the storage of pcs however is a logistical challenge due to the limited 7 day shelf life under standard conditions. during storage, platelets undergo a number of mechanical and biochemical changes contributing to the short shelf life of a pc. these changes are collectively known as the platelet storage lesion. platelet extracellular vesicles (pevs) are known to increase throughout pc storage, due to an increase in platelet activation. as pevs have previously been shown to be pro-coagulant and increase in annexin v binding over pc storage. the aim was to investigate the effect of pc storage time on extracellular vesicle interactions on fibrin clot formation. methods: pcs were sampled on alternate days up to 10 days of storage and centrifuged to achieve acellular plasma. the plasma was subjected to ultracentrifugation (100,000xg) to pellet evs. the size and concentration of evs was assessed using nanoparticle tracking analysis software, followed by a western blot to confirm evs were of platelet origin. the pevs were added at a fixed number to a control pooled plasma sample with added thrombin and tissue plasminogen activator. the time to clot and 50% lysis time were recorded by using the turbidometry of the plasma over time. results: evs isolated from the pc were confirmed to be of platelet origin by western blot using cd41 as a marker of platelet origin and cd9 as an ev marker. pevs caused a significant increase effect on the fibrin clot formation (p < 0.001) when compared to the control plasma. pevs also had a significant effect (p < 0.01) on the fibrinolysis time, extending the time taken to lyse the clot. characterization of mirna from serum derived exosomes in a mouse tibia fracture model of introduction: complex regional pain syndrome (crps) is a debilitating chronic disease that occurs after trauma to the periphery and is intimately associated with nerve injury. its presentation is often described as an injury that is disproportional to the inciting event and manifests neuropathic pain, systemic inflammation, and immune dysregulation. owing in part to our poor understanding of disease aetiology, current treatments for crps are insufficient and as a disease of exclusion there is a lack of quantitative diagnostic markers. exosomes are small extracellular vesicles (sevs) 30-100 nm in size which provide a means of cellular communication through their cargo molecules (protein, mirna, mrna, lipids) , and have demonstrated promise in uncovering mechanisms of disease manifestation and identifying potential diagnostic markers. we have shown previously that crps patients have differential expression of several mirnas in serum derived sevs as compared to healthy controls, but little is known on how this compares to the established mouse tibia fracture model of crps. methods: mice undergoing fracture were anesthetized and subjected to a unilateral tibia fracture followed by casting of the injured limb. after confirming the establishment of pain hypersensitivity, serum samples were collected from fracture model and control mice three weeks post-injury. sevs were isolated by differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna-seq analysis is being performed to identify differentially expressed mirnas. results: nanoparticle tracking analysis showed no significant difference in the number or size of sevs present in the serum from the fracture model and control mice. rna-seq is ongoing and differential mirna expression in sevs from fracture model will be compared to control samples. comparative studies identifying mirnas that are common between crps patients and the rodent model will facilitate the development of correlational outcomes between preclinical and human studies. summary/conclusion: identification of similarities and differences between crps patients and animal models will aid in directing future studies at clinically relevant aspects of crps aetiology and identifying potential diagnostic markers for crps patients. extracellular vesicle-based liquid biopsy in acute myeloid leukaemia: a reliable source of residual disease biomarkers? introduction: acute myeloid leukaemia (aml) is an haematopoietic stem cell disorder with a poor 5-year survival rate. monitoring of measurable residual disease (mrd) in aml patients receiving chemotherapeutic treatment is useful to assess therapy response and predict relapse. indeed, many different leukaemia associated immunophenotypic protein markers (laips) are presently useful to detect mrd. nevertheless, their analysis currently requires invasive bone marrow aspirates, thus severely hindering real-time monitoring of the disease. therefore, alternative peripheral blood-based methods are highly desirable for an easy, real-time and costeffective monitoring of aml progression. this work aims was to assess the feasibility of a peripheral blood ev-based liquid biopsy method for aml disease monitoring, based on the detection of laips with a known negative impact on the prognosis of aml. methods: the profile of evs isolated from 12 paired samples from aml patients' blood plasma collected at diagnosis, complete remission (and some at relapse) was compared and correlated with clinical data. for that, a size-exclusion chromatography (sec) method was optimized to isolate the circulating evs from the blood plasma. the evs of the 12 paired aml patients' blood samples were then characterized according to their size (dls/nta), morphology (tem), proteinto-lipid ratio (lowry/sulpho phosphovanillin assay), surface charge (zeta-sizer) and protein cargo (western blot). results: sec allowed the isolation of size-resolved plasmaderived evs from the peripheral blood of aml patients. isolated evs had a size ranging from 30 nm to 300 nm with an intact morphology, expressing ev-associated markers such as hsp70, cd63, cd81 and cd9. size-resolved evs also had a differential expression of mitofilin, actinin-4, syntenin-1 and annexin-xi proteins. several laips were detected in the isolated evs and their relative abundance changed throughout the stage of the disease. summary/conclusion: our preliminary data shows that aml patients' circulating evs carry relevant immunophenotypic protein markers, which might predict aml clinical outcome. introduction: cell plasticity regulated by the balance between the epithelial-to-mesenchymal transition (emt) and met is critical in the metastatic cascade. extracellular vesicles (evs) may play an important role in this balance by shuttling molecular cargos into recipient cells. this study aims to evaluate the feasibility of profiling mrnas of parental prostate cancer (pca) cells with different phenotypes and their daughter evs using the nanostring low rna input ncounter assay. methods: pc3-epi and pc3-emt cell lines representing epithelial and mesenchymal phenotype, respectively, were generated from original pc3 cell line. the cell culture supernatant was first pre-cleared for any dead cells and debris by centrifugation at 1000 × g for 20 min. without disturbing the pellet, the supernatant was then transferred to a fresh ultracentrifuge tube and centrifuged at 10,000 × g for 20 min at 4°c. the remaining supernatant was then centrifuged to isolate the evs at 100,000 × g for 120 min at 4°c. the evs pellet was further washed in 1× pbs followed by a second centrifugation at 100,000 × g for 120 min at 4°c. the final evs pellet was resuspended in 1× pbs for subsequent characterization (transmission electron microscopy, nanoparticle tracking analysis and western blot) and ncounter assays. the total rna of cells and their daughter evs were assayed by the ncounter pancancer progression panel to determine expression of 770 selected mrnas. the nanostring ncounter low rna input kit with the multiplex 770gene primer pool was used for the pre-amplification of mrna and overnight hybridization with the pancancer progression panel. each sample type was submitted to the assay in biological triplicate. results: when comparing all 12 samples, eisen cluster analysis separated all the cells and all evs into two groups, regardless of their phenotypes. in subgroup analysis, the expression patterns between pc3-epi and pc3-emt cells were significantly different. clec2b, kdr, crip2, il13ra2, cc2d1b were significantly upregulated in pc3-emt cells, while cxcl8, epcam, esrp1, tgfb2, cdh1, s100a14, ovol2 were significantly downregulated in pc3-emt cells. the expression patterns between pc3-epi and pc3-emt evs were also significantly different. tbx1, cav1, col4a1, slc35a3, myc, itgb2, timp4, camk2b, ptgds, p3h2, itgb6, vim, stat3 were all significantly downregulated in pc3-emt cell derived evs. summary/conclusion: the nanostring low rna input ncounter assay can provide reliable mrna expression profiling of evs. the mrna expression patterns are very different between cells and their daughter evs. both cells and evs with different phenotypes have different gene expressions. cancer cell-derived evs containing alphav beta6 integrin regulate cd163, il-6 and il-10 levels in peripheral blood mononuclear cells introduction: extracellular vesicles (evs) mediate communication in the tumour microenvironment and play an important role in cancer progression. previously, we have shown the enrichment of alphav beta6 integrin in small extracellular vesicles (sevs) isolated by differential ultracentrifugation and iodixanol density gradient from pc3 prostate cancer cells. we have also shown in the past that alphav beta6-positive sevs induce peripheral blood mononuclear cell (pbmc) polarization by increasing the expression of pro-tumorigenic m2 markers, such as cd163 and cd204. finally, we have demonstrated that down-regulation of alphav beta6 integrin up-regulates the stat1-interferon stimulated genes (isgs) pathway in cancer cells and in sevs released by them. methods: in order to investigate whether prostate cancer cell-derived vesicular stat1 has a causal effect in pbmc polarization, we down-regulated alphav beta6 and stat1 in prostate cancer cells derived sevs using sirna as well as crispr-cas9 strategies. the sevs isolated from these cells were used to analyse m2 polarization by measuring the levels of cd163 in pbmc. the results show that sevs lacking alphav beta6 inhibit cd163 levels in pbmc in a stat1-independent manner. analysis of cytokines released by pbmc upon incubation with sevs lacking alphav beta6, show that pbmc selectively up-regulate the levels of il-10 and il-6, which are predominantly anti-tumorigenic cytokines. in contrast, sevs lacking alphav beta6 do not upregulate pro-angiogenic cytokines, such as vegf. summary/conclusion: these findings suggest that cancer cell-derived sevs containing alphav beta6 integrin promote a pro-tumorigenic pbmc phenotype in the tumour microenvironment by regulating cd163, il-6 and il-10 levels. introduction: the recognition of donor-mhc molecules by recipient t cells triggers the immune response leading to rejection of allografts. our recent studies have documented the presence of high numbers of recipient apcs displaying donor-mhc molecules (cross-dressed) on their surface in the lymphoid organs of mice after skin, heart or pancreatic islet transplantation. in addition, we have reported that acquisition of allogeneic mhc molecules by host apcs (mhc crossdressing) is mediated by donor-derived extracellular vesicles (evs) trafficking through blood and lymphatic vessels (marino et al. science immunology, 2016) . in the present study, we investigated the ability of allogeneic evs and allo-mhc-cross-dressed cells to initiate a t cell alloresponse in vitro and in vivo. methods: evs were isolated (using differential centrifugation) from balb/c bone marrow derived dendritic cells (bmdcs). these evs were used to cross-dress b6 splenocytes in vitro. the transfer of donor mhc class i and ii on b6 cells was analysed by imaging flow cytometry. next, t cells from b6 mice were cultured in vitro with either allogeneic bmdc-derived balb/c evs or b6 spleen cells crossdressed with allogeneic balb/c mhc. alternatively, 2 × 109 balb/c or b6 bm derived evs or 20 × 106 balb/c bm cells were injected iv to b6 mice. in both cases, the t cell response was assessed by activation markers detection, infg production and cell proliferation. results: apcs cross-dressed with allogeneic mhc molecules can trigger a pro-inflammatory direct alloresponse by t cells in vitro and in vivo. on the other hand, allogeneic evs alone were only able to induce early t cell activation but not proliferation in vitro. furthermore, injection of mice with allogeneic evs alone could induce some but suboptimal alloresponse in vivo and only when administered with complete freund's adjuvant. summary/conclusion: blocking donor evs release and subsequent recipient apc cross-dressing may represent a promising target to selectively inhibit anti-donor t cell inflammatory responses thus achieving long-term allograft survival. funding: r01dk115618. antifungal antibiotic activity of outer membrane vesicles from adherent lysobacter enzymogenes c3 against therapeutic and biocontrol targets. rutgers university, new brunswick, usa introduction: lysobacter enzymogenes is a predatory gram negative bacterial species being studied for biocontrol activity against fungi. planktonic l. enzymogenes c3 produces outer membrane vesicles (omv) harbouring small molecule antifungal antibiotics (meers et al. 2018) . we show here that the more biologically relevant surface-associated c3 exerts remote antifungal activity via omv as well. the results have important consequences regarding the natural mechanism of biocontrol of fungal pathogens by c3 as well as isolation and delivery of therapeutically relevant antifungal compounds. methods: omv were isolated from scraped adherent c3 culture on agar by similar methods to meers et al 2018. omv were stained in some cases with fluorogenic syto 9 dna stain for microscopic observation. fungal growth was monitored via turbidity readings in liquid culture or photomicrographs on agar. c3 was also grown on polycarbonate filter membranes with defined pore sizes to monitor growth of fungal cells on the opposite side. vesicles were also labelled with an amine-reactive probe alexa-555 and washed 4x by sedimentation. binding of labelled omv to fungal cells was observed by epifluorescence microscopy. results: syto 9-stained vesicles from surface-adherent c3 were similar to previously observed~130 nm vesicles (meers et al., 2018) . the isolated vesicles inhibited growth of saccharomyces cerevisiae or candida albicans in liquid cultures at similar potency and were active against the filamentous species fusarium subglutinans grown on agar or maize leaves. c3 cultures grown on filters with 400 nm pore size but not 30 nm were able to inhibit the hyphal growth of f. subglutinans on the opposite side. similarly c3 on filters with a 200 nm pore size were able to inhibit growth of c. albicans. observation of fluorescently-labelled c3 omv after interaction with c. albicans showed binding specifically to hyphae or pseudohyphae and for f. subglutinans to the growing hyphal tips. summary/conclusion: the omv of c3 specifically bind and inhibit the growth of fungal hyphae of various species without direct c3 cell contact. these data elucidate mechanisms of biocontrol and suggest strategies for production of therapeutic antifungal antibiotics. meers et al. elucidating the cellular uptake and tissue distribution mechanism of cell derived vesicles, a novel therapeutic carrier hui-chong lau a , jae young kim a , jin-hee park a , jun-sik yoon a , min jung kang a and seung wook oh b a mdimune inc, seoul, republic of korea; b mdimune inc, seattle, usa introduction: cell derived vesicles (cdvs) are emerging as a novel therapeutic carrier. one of the crucial factors in the development and therapeutic applications of cdvs is to understand the precise mechanism by which vesicles find and enter the target cells. in this study, we aim to investigate the uptake mechanism of cdvs produced from natural killer (nk) cells using a manufacturing process established at mdimune inc. both in vitro uptake assay and in vivo distribution analysis were performed to provide precise insights into how cdv exert its effect at the cellular level. methods: nk cells were mainly used to produce cdvs. breast cancer cells, bt549, and human and rodent endothelial cells, with a varying degree of icam-1 expression, were used to determine the effect of lfa-1 expressed on the surface of nk-cdvs in cellular uptake using facs and confocal imaging analysis. next, various inhibitors for uptake pathways, such as phagocytosis, dynamin dependent endocytosis, and receptor mediated endocytosis, were used to understand the underlying mechanism of cellular uptake of cdvs. biodistribution profile of cdvs were characterized using both normal and tumour xenograft models by ivis imaging. results: using a recently established manufacturing process, we demonstrate that nk-cdvs can efficiently enter the target cells. this study also shows that the cellular uptake depends on the molecular interaction between icam-1 and lfa-1. in vivo distribution profile of nk-cdvs are also assessed using various tumour models. furthermore, we present a cellular uptake mechanism involved in the entrance of cdvs into the target cells. summary/conclusion: this study demonstrates that the cdvs produced at the manufacturing scale can be easily taken up by cells via specific cellular pathways. this finding will facilitate the development of more efficient therapeutics for cancer and other debilitating diseases. myofibroblasts-derived microvesicles increase dermal fibroblasts collagen production through plgf-1 syrine arif, sebastien larochelle and véronique j. moulin chu de québec -université laval, loex, québec, canada introduction: a proper wound healing of the skin involves angiogenesis, extracellular matrix (ecm) remodelling and re-epithelialization. these three mechanisms require well-organized interactions between different cell populations. a key role in this context is played by myofibroblasts (wmyo), a cell population mainly differentiated from dermal fibroblasts. these cells contract wound edges and synthesize new ecm. we previously showed that myofibroblasts predominantly produces microvesicles (mvs) and can favour angiogenesis. however, proteomic analysis of mvs from our previous studies indicated some molecules that can potentially be implicated in ecm remodelling. in this study, we evaluated whether myofibroblasts-derived mvs could affect dermal fibroblasts who are highly responsible for ecm regulation. methods: mvs were isolated by differential centrifugation of medium collected from wmyo cells. number and size of mvs were characterized by transmission electron microscopy and nanosizer. multiplex assays of 45 cytokines were evaluated in mvs samples, wmyo and mvs-depleted medium. to examine the interaction of mvs with fibroblasts, we evaluated the uptake of mvs isolated from wmyo transduced with a fluorescent protein. we then treated fibroblasts cultures with mvs or a selected cytokine for 5 days and evaluated collagen production. lastly, we neutralized the selected cytokine in mvs samples before evaluating collagen production. results: plgf-1 was the cytokine detected in mvs samples in large amount (0.88 ± 0.63 pg/µg proteins in mvs). fibroblasts treated with mvs or plgf-1 significantly stimulated pro-collagen i level production with a fold change of 1.80 ± 0.18 and 2.07 ± 0.18. moreover, the neutralization of plgf-1 present in mvs significantly inhibited the production of pro-collagen i by dermal fibroblasts. summary/conclusion: our results indicated that mvs influence fibroblasts pro-collagen production through plgf-1 signalling. funding: this work was supported by natural sciences and engineering research council of canada (nserc) (rgpin2014-04404); les fonds de recherche du québec-santé (frqs) via the research centre funding grant; the quebec cell and tissue and gene therapy network-thécell (a thematic network supported by frqs). structural insights on fusion mechanisms of extracellular vesicles with model plasma membranes introduction: extracellular vesicles (evs) represent a potent intercellular communication system. while their functional biological properties are more and more investigated, the biophysical aspects of their interaction with recipient cells are often overlooked. small size (30 to a few hundred nanometres in diameter) of evs and their heterogeneous origin still pose a great challenge for their isolation, quantification and biophysical/biochemical characterization. in particular the complex network of interactions between differently classified evs and recipient cells remains to be further revealed. here we deeply investigate the fusion mechanism between evs and a model plasma membrane system by an interplay of different structural/morphological techniques to get a molecular description of the interaction helping to clarify the role of different membrane compartments on the evs uptake mechanism. standardized protocols and good manufacturing practice conditions were employed to derive highly stable vesicles of defined size and reproducible molecular profiles from umbilical cord multipotent mesenchymal stem (stromal) cells. after a thorough biophysical and biochemical characterization of evs non-contact liquid imaging atomic force microscopy (afm) and, in parallel, neutron reflectometry (nr), as well as small angle neutron scattering (sans) experiments were performed on evs to determine their interaction with model plasma membranes in the form both of supported lipid bilayers and suspended unilamellar vesicles of variably complex composition. results: we observed that evs tend to fuse with the model membranes with a preferential interaction with the external layer of the fluid membrane. moreover we revealed a stronger interaction with the liquid ordered domains, strengthening the hypothesis of a critical role of lipid rafts in fusion mechanisms. summary/conclusion: our results on the analysis of the interaction of evs with artificial lipid membranes could provide insights on the internalization mechanisms of evs. the approach shown here can be further extended to convey incremental complexity, adding glycolipid and membrane proteins to the model lipid bilayers. this approach combined with data on the specific biological function of each ev subpopulation as retrieved by standard functional assays, will turn useful to select the crucial molecular aspects of evs internalization by cells. introduction: platelet-derived extracellular vesicles (pev) are the most abundant circulating extracellular vesicle (ev) and exhibit platelet-like properties, hence the original term "platelet dust". direct phenotyping of ev surface markers within biofluids is challenging often requiring time-intensive purification steps that can significantly alter resultant ev population characteristics. the exoview™ (nanoview biosciences) specifically captures ev sub-populations and was used to characterise the ev content of platelet free plasma (pfp) and a potential novel haemostatic agent designed for the treatment of severe trauma and haemorrhage, platelet enhanced plasma (pep). methods: freeze-thaw cycling of platelet rich plasma/ expired platelet concentrates was followed by centrifugation to remove platelet remnants and yeilded pep. pfp controls were prepared by double centrifugation (2000 g for 20 minuntes followed by 13,000 g for 2 minutes). rotational thromboelastometry (rotem) and calibrated automated thrombography (cat) were used to assess ev driven haemostasis and thrombin generation. a dilutional and hypothermic model of coagulopathy was designed to assess pep. ev capture arrays comprised of anti-cd41, anti-cd63, anti-cd81 and anti-cd9 were used (exoview™, nanoview biosciences). captured vesicles underewent interferometric imaging and were quantified, sized and further probed with fluorescent tetraspanin markers, annexin-v and intravesicular markers. results: pep is highly procoagulant, exhibits enhanced thrombin generation and can restore haemostasis in a dilutional model of coagulopatic whole blood. pep can be generated from expired platelet concentrates, potentially allowing for upscalable production. the predominant vesicle population were pev with a large cd41/cd9 population that contained a smaller subpopulation of phosphatidyserine positive procoagulant vesicles. pfp as expected has a much lower number of pev and a cd81 positive ev population. summary/conclusion: pep is a unique resuscitation fluid containing high pev levels for the potential treatment of severe trauma and haemorrhage. exoview measurements can be performed in unpurified plasma and may be useful for measuring circulating ev in health and disease. funding: defence and security accelerator, dstl therapeutic effect of exosomes in mice model of autism daniel offen a , reut horev a , nisim perets a , ehud marom b , uri danon b and yona gefen b a tel aviv university, tel aviv, israel; b stem cell medicine ltd., jerusalem, israel introduction: during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. we have previously shown that intranasal administration of msc-exo, cross the bbb and significantly ameliorate autistic-like behavioural phenotype in btbr and shank3 animal models of autism, representing a potential therapeutic strategy to reduce symptoms of autism spectrum disorder (asd). our objective is to study the mechanism of action and the cellular pathways in which the msc-exo activate their target, we performed rna sequencing analysis of primary neurons isolated from shank3 mice treated with msc-exo. methods: primary neuronal cell cultures were prepared from newborn shank3 homozygotes mice model of autism. cultures were treated with msc-exo (10^7 particles/ul), isolated from human adipocytes, followed by rna sequencing. the alterations in gene expression between the treated and intact neurons were analysed for gene ontology and pathways and were also compared to proteomics analysis of the msc-exo in order to find regulatory proteins that may lead to these differences. results: bioinformatic analysis revealed several up-regulators proteins that might be responsible for the increase in anti-inflammatory and protective factors seen in the mice neurons treated with msc-exo. one of them is bdnf which is known as an essential growth factor responsible for neuroprotection and neurogenesis. importantly, no difference in the genetic expression of cancer-related genes was identified following msc-exo treatment indicating for their safety. summary/conclusion: our data suggest that adipocytederived msc-exo carry therapeutic potential in asd via alternation in gene-expression related mainly to immuno-modulation, reduce neuroinflammation and increase neuroprotection and neurogenesis. the beneficial effects of the exosomes treatment in mice models is being translated into a novel, easy to administer, a therapeutic strategy to reduce the symptoms of asd. introduction: autologous blood-derived products gain increasing focus in regenerative medicine, especially in orthopaedics and osteoarthritis therapy. this disease is characterised by cartilage degradation and inflammation among other symptoms, which are targeted by conventional therapies, but genuine cartilage regeneration is rarely achieved. citrate-anticoagulated platelet rich plasma (cprp) is often clinically applied to stimulate soft and hard tissue healing. recently, cell-free alternatives to cprp including hyperacute serum (hypact™ serum) have been developed. cprp and hypact™ serum contain specific profiles of growth factors, however, they also contain extracellular vesicles (evs) that harbour signal molecules including mirna. methods: evs were enriched by ultracentrifugation (uc) followed by size exclusion chromatography (sec) to obtain purified evs. particle size and concentration of each fraction was measured by nanoparticle tracking analysis (nta). fractions with the highest amount of particles were pooled and concentrated via uc, before mirna expression was assessed via screening with a panel of 372 mirna-specific primer pairs by rt-qpcr. presence of evs was confirmed by cryoelectron microscopy. results: the ev concentration tended to be lower in hypact™ serum than in cprp as determined via nta. similarly, lower diversity of mirna species was found in hypact™ serum than cprp evs. around 90% of detected mirnas were found in both blood products, whereas only 30% of mirnas were shared between evs from cprp and hypact™ serum. while mirnas such as mir-101 were consistently depleted in evs compared to the corresponding blood product, others like mir-27a were in enriched in hypact™ evs, but not cprp evs, indicating release of specific mirnas via evs in response to clotting. summary/conclusion: although the purification resulted in high loss of evs, we identified specific mirnas enriched in evs from cprp and hypact™ serum. their functional spectrum with respect to osteoarthritis therapy focuses on inhibition of inflammation, inhibition of tissue remodelling via matrix degrading enzymes as well as preventing senescence. this renders blood product derived evs as interesting candidates for in vitro and in vivo testing with respect to cartilage regeneration. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst3-f-5030664/003-2017. protective role of shiitake mushroom-derived exosome-like nanoparticles in d-galactosamine and lipopolysaccharide-induced acute liver injury in mice baolong liu, xingyi chen and jiujiu yu university of nebraska lincoln, lincoln, usa introduction: fulminant hepatic failure (fhf) is a rare, life-threatening liver disease with poor prognosis. new therapeutic interventions are urgently needed to treat this disease. administration of d-galactosamine (galn) and a low dose of lipopolysaccharide (lps) triggers acute liver damage in mice, which simulates many clinical features of fhf in humans and therefore is widely used to investigate the molecular mechanisms and potential therapeutic interventions of fhf. recently, suppression of the nucleotide binding domain and leucine rich repeat related (nlr) family, pyrin domain containing 3 (nlrp3) inflammasome was shown to alleviate the severity of lps/galn-induced liver injury in animal models. therefore, the goal of this study was to identify food-derived exosome-like nanoparticles (elns) with anti-nlrp3 inflammasome function to potentially control fhf. methods: seven commonly consumed mushrooms were used to extract elns, which were examined for anti-nlrp3 inflammasome activities in primary macrophages. results: it was found that these mushrooms contained elns composed of biomolecules including rnas, proteins, and lipids. among these mushroom-derived elns, only shiitake mushroom-derived elns (s-elns) strongly inhibited nlrp3 inflammasome activation by blocking the inflammasome assembly. this inhibitory effect was specific for the nlrp3 inflammasome because s-elns had no impact on activation of the absent in melanoma 2 (aim2) inflammasome. s-elns also inhibited the secretion of interleukin (il)-6 and both protein and mrna levels of the il1b gene in macrophages. remarkably, pre-treatment of s-elns protected mice from lps/galn-induced acute liver injury. summary/conclusion: therefore, s-elns, identified as potent inhibitors of the nlrp3 inflammasome, represent a new class of agents with the potential to combat fhf. approaches to assess clinically available exosomes' quality and safety introduction: recent adverse events resultant from an exosome product use in a nebraska clinic, highlight the importance of assuring product quality and safety standards. an often-overlooked safety risk is ancillary reagents remaining within a finished product. when processes to obtain exosomes utilize cow proteins such as fbs or bovine sera albumin, failure to adequately remove these can result in significant adverse allergic reactions. we evaluated 3 different exosome products to test the hypothesis that purity of some products may not be consistent with actual product quality and safety profiles claimed. methods: three different exosome products (manufacturer a, b, and c) were prepared per their instructions for use. sample source identity was blinded from assaying scientists. an independent cro service was used to conduct the experiments to ensure unbiased assay execution and data collection. exosome suspensions were sampled undiluted for bovine protein content using commercially available bovine secretome protein arrays from ray biotech. a total of 30 different proteins found in bovine serum were quantified. results: six of 30 proteins were not detected in any sample. 8 of 24 array antibodies were found to cross react with human antigens. of the 16 bovine proteins that were acceptable for analysis, manufacturers a, b, and c exosomes contained 15 of 16 proteins,13 of 16 proteins, and 0 of 16 proteins, respectively. concentrations of individual bovine proteins ranged from 0.2 to 1,220.1 ng/ml. summary/conclusion: these results indicate manufactures a and b are selling potentially dangerous products. the successful implementation of exosome products into the clinic requires equivalent demonstrations of safety and quality. this requires adopting strict quality standards and safety testing during their production. physicians must require safety data prior to clinical use. engineering pro-healing ev cargo using a closed-system bioreactor. introduction: chronic wounds, including diabetic ulcers and pressure ulcers, are difficult and expensive to treat. while tissue engineering approaches have largely failed as a viable treatment for chronic wounds, we hypothesize that stem cell-derived extracellular vesicles (evs) may provide several unique advantages. zenbio, inc has developed a methodology to generate commercial-scale stem cell-derived exosomes using a closedsystem hollow fibre bioreactor capable of continuous ev production. additionally, we have shown that by manipulating the cellular environment, we can improve the pro-healing capacity of the evs.this technology leverages the complex healing capabilities of stem cells without the obstacles of replicating cells. methods: we have demonstrated that a mild heat shock resulted in evs enriched for stress-response proteins and increased pro-healing activities in vitro. we extended this innovative approach to include stimulating adipose stem cells with combinations of heat shock and growth factors to generate differential extracellular vesicle packaging that enhances pro-healing activity. to monitor reproducibility across lots and batches, we rigorously characterized tuned evs for particle size and number as well as surface marker and cargo composition. results: our results using tuned evs showed efficacy using cellular models of inflammation, motility, vascularization, collagen production and metalloprotease activity. we utilized an established murine model of pressure ulcers to assess the in vivo efficacy of the tuned evs. these studies showed a single injection into the wound site activated a more rapid wound closure, increased collagen deposition and reduced dermal thickness compared to saline control. summary/conclusion: these data strongly support our hypothesis that evs may be selectively modified to improve their wound healing activity by modulating the culture or tissue microenvironment. future studies will use chronic wound models to determine optimal dosing and routes of administration. introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) can reduce inflammation, promote healing and improve organ function thereby providing a potential "cell-free" therapy. prior to clinical translation, there is a critical need to synthesize existing preclinical evidence supporting their efficacy. this systematic review provides the most comprehensive evidence map of methods, safety and efficacy for msc-ev research to date. methods: medline and embase were systematically searched for in vivo interventional studies using msc-evs. two reviewers extracted data for: 1) methodology, 2) study design, 3) intervention details and 4) efficacy/ adverse events. results: after screening 754 articles, 206 studies met our eligibility criteria. msc-evs were used to treat a variety of diseases including renal (16%), neurological (12%) and cardiac (10%) conditions. benefits were described in 95% of studies across all organ systems and adverse effects were seen in only three studies; two showing tumour growth. however, several key methodological concerns were evident. based on size criteria for ev subtypes (exosomes/small evs~30-150 nm, microvesicles~150-1000 nm) only 60% of studies used appropriate nomenclature. ultracentrifugation (70%) and isolation kits (23%) were the most common isolation methods despite marked differences in yield and purity. evs were inconsistently dosed by protein (68%), particle number (16%) or cell count (4%), hindering inter-study comparisons. two-thirds of studies used xenogeneic evs suggesting immunocompatibility. techniques to determine size, protein markers and morphology was highly heterogeneous, and only 12 and 4 studies met the characterization standards recommended in the misev 2014 and 2018 guidelines, respectively. finally, 50% of studies did not incorporate randomization which represents a high risk for bias and only a quarter performed biodistribution studies. summary/conclusion: this systematic review reveals extensive heterogeneity in methods and intervention details for animal studies of msc-evs. nonetheless, nearly all studies showed significant benefits in a wide range of distinct conditions. the knowledge gaps we identified highlight important opportunities for improving preclinical design and the need for more standardized approaches in this growing field of ev therapeutics. msc-exosomes as next generation therapeutics for atopic dermatitis exocobio inc, seoul, republic of korea introduction: atopic dermatitis (ad) is a systemic inflammatory disease with unknown cause. recent approval of a targeted therapy, dupilumab, opens new era of ad management. however, current therapeutic options for ad are only targeting inflammation, a component of ad vicious cycle including itching and barrier disruption. human mesenchymal stem cells (mscs) have been highlighted as a novel therapy for suppressing allergic progress of ad in clinical studies. unfortunately, phase iii clinical study of human umbilical cord blood mscs for ad was failed with unknown reason. previously, our group reported that exosomes derived from human adipose tissue-derived mscs (asc-exosomes) alleviated the pathological symptoms in a murine ad model with concomitant reduction of inflammation. methods: our group has further investigated the therapeutic effects of human asc-exosomes in an alternative murine ad model with skin barrier defects. large scale isolation of asc-exosomes was performed by tangential flow filtration and isolated asc-exosomes were characterized according to the recommendation by the isev. the protein and lipid cargo were also analysed. results: we found that asc-exosomes induced restoration of skin barrier by inducing de novo lipid synthesis and reduced the levels of multiple inflammatory cytokines. in addition, asc-exosomes suppressed the expression of itching-causing cytokines. transcriptomic analysis of ad skin lesions revealed that asc-exosomes reversed the abnormal expression of genes functioning in skin barrier function, lipid metabolism, and cell cycle. summary/conclusion: taken together, asc-exosomes could be a promising cell-free therapeutic option for the treatment of ad, which affecting inflammation, skin barrier function, and itching. cell derived vesicles: unravelling the science of novel vesicles with therapeutic promises introduction: cell derived vesicles (cdvs) are nanosized vesicles produced by serially extruding cells through small pores. a growing number of studies have implicated their therapeutic potentials, with superior yield compared to other extracellular vesicles (evs). however, two key objectives remain to be accomplished to demonstrate the utility of cdvs in clinical applications. first, a manufacturing process has to be developed to allow a large-scale production of cdvs. next, these novel vesicles need to be thoroughly characterized at multiple levels. methods: manufacturing-scale extruders were developed to allow extrusion of large volume of cell suspension in a single process. cdvs with approximately 150-200 nm in diameter were obtained by a serial extrusion. crude samples were then purified using the tangential flow filtration method to further remove cellular impurities. finally, physical and biochemical characteristics of purified cdvs were analysed using dls, nta, cryo-em, and facs analysis. additionally, cdvs were subject to multi-omics profiling to comprehend our understanding in molecular contents of cdvs. both mesenchymal stem cells (mscs) and natural killer (nk) cells were used for this study. results: in this study, we first demonstrate that the large-scale extruder efficiently produce cdvs with consistent quality at the scale that are compatible for clinical applications. surface marker and membrane composition analyses show that the cdvs are primarily formed using plasma membrane of source cells, with characteristic cellular markers enriched on the surface. comprehensive profiling of molecular components reveals the unique properties of cdvs as well as the underlying mechanism of formation of cdvs. summary/conclusion: recently, we have established a manufacturing process to enable clinical applications of cdvs. this study also highlights key molecular features of cdvs that can be harnessed to offer a powerful tool for regenerative and anticancer medicine. antifibrotic properties of extracellular vesicles derived from human induced pluripotent stem cells introduction: fibrosis is a pathological condition resulting from abnormal healing of various tissues. it is triggered by activation of fibroblasts and their subsequent transition to myofibroblast. in consequence, excessive deposition of extracellular matrix proteins leads to impaired organ function. to revert this process, we employed extracellular vesicles (evs) derived from human induced pluripotent stem cells (hipscs). as a model system, we used human cardiac fibroblasts (hcfs), since heart fibrosis constitutes a serious socioeconomic problem worldwide. methods: we isolated evs from conditioned media from three hipsc lines using ultrafiltration combined with size exclusion chromatography methods. next, we analysed the evs by nanosight, transmission electron microscopy, mass spectrometry and western blot methods. finally, we treated tgf-b-stimulated hcfs with hipsc-evs and evaluated expression of fibrosisrelated genes using real-time qpcr, western blot and fluorescence microscopy. results: we detected anti-fibrotic properties of hipsc-evs exerted on hcfs pre-stimulated with tgf-b. the evs significantly decreased the expression levels of acta2, fn, tnc, snai2, col1a1 and reduced the number of myofibroblasts. the canonical profibrotic tgf-b-dependent smad2/3 pathway was significantly attenuated in response to ev-treatment. summary/conclusion: in this study we demonstrated strong anti-fibrotic function of hipsc-evs. our findings can further be exploited for future medical applications to treat fibrotic diseases, such as heart fibrosis. funding: this work was supported by the project sonata12: umo-2016/23/d/nz3/01310 from the national science centre of poland to sbw. induced pluripotent stem cells-derived extracellular vesicles ameliorates d-galactosamine and lipopolysaccharide induced acute liver failure tianjin third central hospital affiliated to nankai university, tianjin, china (people's republic) introduction: liver failure is among the most causes of death in patients with liver disease. promoting liver regeneration will help patients with liver failure recover on their own. extracellular vesicles (evs) can released by induced pluripotent stem cells (ipscs) through paracrine effects and play a pivotal role in inter-cellular communication in the treatment of disease. in this study, we investigated whether the ipscs-evs have therapeutic effects on acute liver failure. methods: the ipscs-evs were isolated by ultracentrifugation and identified using nanoparticle tracking analysis, transmission electron microscopy and western blotting. the isolated ipscs-evs were administrated d-galactosamine-injured heprg cells in vitro and tail intravenously injected into d-galactosamine and lipopolysaccharide induced acute liver failure model mice in vivo, respectively. the anti-apoptosis role and potential mechanism were evaluated using flow cytometry and immunofluorescence staining. and alanine transaminase (alt) and aspartate transaminase (ast) in serum, h&e staining and tunel staining were explored the effect of ipscs-evs on liver injured and liver function. finally, high throughput sequencing of small rnas was performed to investigate mirna expression profiles in ipscs-evs and ipscs. results: the ipscs-evs that were all 50-200 nm, doublelayered and oval or round cellular vesicles and expressed the marker proteins cd63, tsg101 and hsp70. in vitro, the ipscs-evs treatment inhibited heprg apoptosis induced with d-galactosamine in a time-and dosedependent manner and promote the proliferation of hepatic stem cells. in vivo results showed that ipscs-evs significantly alleviated liver failure, improved liver function and prolonged the survival period. tunel assay showed that ipscs-evs suppress apoptosis of hepatocytes. moreover, mirna expression profiles analysis found that mir17-92a cluster and mir302-367 cluster were enriched in ipscs-evs and ipscs. summary/conclusion: these findings indicated that ipscs-evs could ameliorate d-galactosamine and lipopolysaccharide induced acute liver failure to attenuate hepatocyte apoptosis, which will be benefit for therapy of liver disease in the future. msc-derived extracellular vesicles promote human cartilage regeneration by control of autophagy introduction: osteoarthritis (oa) is a rheumatic disease leading to chronic pain and disability with no effective treatment available. recently, allogeneic human mesenchymal stromal/stem cells (msc) entered clinical trials as a novel therapy for oa. increasing evidence suggests that therapeutic efficacy of msc depends on paracrine signalling. here we investigated the role of bone marrow msc-derived extracellular vesicles (bmmsc-evs), an important component of msc secretome, in cartilage repair. methods: to test the effect of bmmsc-evs on oa cartilage inflammation the tnf-alpha-stimulated human oa chondrocytes were treated with bmmsc-evs and inflammatory gene expression was measured by qrt-pcr after 48 h. to access the impact of bmmsc-evs on cartilage regeneration the bmmsc-evs were added to the regeneration cultures of oa chondrocytes, which were analysed after 4 weeks for glycosaminoglycan content by dmmb and qrt-pcr. paraffin sections of the regenerated tissue were stained for proteoglycans (safranin-o) and type ii collagen (immunostaining). results: we show that bmmsc-evs promote cartilage regeneration in vitro. treatment of oa chondrocytes with bmmsc-evs induces production of proteoglycans and type ii collagen and promotes proliferation of these cells. msc-evs also inhibit the adverse effects of inflammatory mediators on cartilage homoeostasis. our data show that bmmsc-evs downregulate tnfalpha induced expression of pro-inflammatory cox-2, pro-inflammatory interleukins and collagenase activity in oa chondrocytes. the anti-inflammatory effect of bmmsc-evs involves the inhibition of nfκb signalling, activation of which is an important component of oa pathology. autophagy, a cellular homoeostatic mechanism for the removal of dysfunctional cellular organelles and macromolecules, is essential to maintaining chondrocytes survival and differentiation. the expression of autophagy regulators is reduced in osteoarthritic joints, which is also accompanied by increased chondrocyte apoptosis. our preliminary data indicate that bmmsc-evs carry mrna of natural autophagy inducers and promote autophagy in oa chondrocytes. therefore, we hypothesize that msc-evs exert their beneficial effects on cartilage regeneration by restoring the expression of autophagy regulators. summary/conclusion: in summary, our findings indicate that bmmsc-evs have ability to promote oa cartilage repair by reducing the inflammatory response and stimulation of oa chondrocytes to produce extracellular matrix, the essential processes for restoring and maintaining cartilage homoeostasis. thus, msc-evs hold great promise as a novel therapeutic for cartilage regeneration and osteoarthritis. large-scale preparations of small extracellular vesicles from conditioned media of mesenchymal stromal cells modulate therapeutic impacts on a newly established graft-versus-host-disease model in batch dependent manners introduction: extracellular vesicles (evs) harvested from supernatants of humane adult bone marrow-derived mesenchymal stem/stromal cells (mscs) can suppress acute inflammatory cues in a variety of different diseases, including graft-versus-host disease (gvhd) and ischaemic stroke. furthermore, they can promote regeneration of affected tissues. following a successful clinical treatment attempt of a steroid refractory gvhd patient, we intend to optimize msc-ev production strategies for further clinical applications. as we observed functional differences of independent msc-ev preparations in vitro, we aimed to adopt an in vivo gvhd model for the more advanced functional testing of different msc-ev preparations. methods: to this end we set up a bone marrow transplantation mouse model in which endogenous bone marrow was myeloablated by ionizing irradiation (iir). gvhd was induced by the transplantation of major histocompatibility mismatched allogeneic spleen-derived murine t cells. if not treated otherwise, myeloablated mice developed severe gvhd symptoms. results: the gvhd symptoms were effectively suppressed, when msc-ev preparations were applied at 3 consecutive days, which exerted immune modulatory effects in a mixed-lymphocyte reaction assay. msc-ev preparations lacking in vitro immune modulating activities, however, hardly improved the symptoms of the gvhd mice. thus, our results demonstrate that not all msc-ev preparations harvested from adult bone marrow-derived mscs contain the same therapeutic potential. summary/conclusion: thus, successful transplantation of msc-evs into the clinics requires a platform allowing identification of msc-ev preparations with sufficient therapeutic, most probably immune modulating activities. funding: this research was funded by sevrit leitmarkt lifescience.nrw ls-1-1-051 g. introduction: malnutrition impacts approximately 50 million children worldwide and is linked to 45% of global mortality in children below the age of five. severe acute malnutrition (sam) is associated with intestinal barrier breakdown and epithelial atrophy. extracellular vesicles including exosomes (evs; 30-150 nm) can travel to distant target cells through biofluids including milk. since milk-derived evs are known to induce intestinal stem cell proliferation, this study aimed to examine their potential efficacy in improving malnutrition-induced atrophy of intestinal mucosa and barrier dysfunction. methods: mice were fed either a control (18%) or a low protein (1%) diet for 14 days to induce malnutrition. from day 10 to 14, they received either bovine milk evs enriched using differential ultracentrifugation and sucrose gradient purification or control gavage and were sacrificed on day 15, 4 hours after a fluorescein isothiocyanate (fitc) dose. tissue and blood were collected for histological and epithelial barrier function analyses. results: mice fed low protein diet developed intestinal villus atrophy and barrier dysfunction. despite continued low protein diet feeding, milk ev administration improved intestinal permeability, intestinal architecture and cellular proliferation. summary/conclusion: our results suggest that evs enriched from milk should be further explored as a valuable adjuvant therapy to standard clinical management of malnourished children with high risk of morbidity and mortality. funding: cb was generously awarded a catalyst grant from the centre for global child health at the hospital for sick children to support this work. the impact of spheroids culture on mesenchymal stem cells and ev production introduction: mesenchymal stem/stromal cells (mscs) are now widely believed as bio-factories releasing bioactive products responsible for their therapeutic effect, i.e. cytokines, chemokines, and extracellular vesicles (evs). mscs are highly sensitive to physical stimuli from their surrounding microenvironment and can change their characteristics in response to their environment. the application of 3d spheroids cell culture allows mscs to adapt to their cellular niche environment which, in turn, influences their paracrine signalling activity. we aim to determine how 2d and 3d culture microenvironments can modulate the ev production and investigate their anti-fibrotic activity. methods: for 2d culture, bone marrow-derived mscs were cultured on standard tissue culture plastic. for 3d culture, mscs were aggregated into spheroids using non-adherent 96-well plates and cultured with addition of 0.25% methylcellulose. to collect conditioned media, both 2d and 3d mscs were cultured using serum free medium for 4 days. evs were isolated by serial ultracentrifugation and were characterised on exoview platform which allows simultaneous detection of particle size and expression of cd81/cd63/cd9. cell lysates were collected for mirna isolation and qrt-pcr was performed to analyse expression of candidate mirnas. to model the progress of lung fibrosis, human lung fibroblasts (hlfs) were cultured with tgf-β1 to induce fibroblast activation, subsequently exposed to 2d and 3d evs, and collagen production was measured. further, 2d and 3d msc-evs were added into human lung mscs isolated from healthy and ipf patients and cell proliferation was assessed using mts assay. results: 2d and 3d msc-evs have similar ev characteristics in terms of particle size and ev tetraspanin markers expression. exoview analysis showed expressions of cd81/cd63/cd9 and average particle diameters of <100 nm. on a cellular level, we identified a panel of anti/pro-fibrotic mirnas which are differentially expressed in 2d and 3d mscs. 2d and 3d msc-evs have similar anti-fibrotic activity shown by their ability to reduce collagen deposition in hlf cultures. both 2d and 3d msc-evs could promote cell proliferation on ipf lung mscs but no overall effect on healthy lung mscs. summary/conclusion: this concept of engineering the cellular microenvironment to promote ev production is as yet untouched and we foresee that in 3d cultures, we can culture mscs for longer timeframe and therefore maximising the overall ev production process. the outcome presents future potential for 3d culture of msc to increase the efficiency and feasibility of scalable ev production. outer membrane vesicles from photobacterium damselae subsp. piscicida: characterization and antigenic potential introduction: photobacterium damselae piscicida (phdp) is a gram-negative bacterium that causes a septicaemia in > 20 fish species worldwide. it represents a major drawback for aquaculture, whose importance has been sharply growing as a food supplier. given the phdp massive mortality and widespread antibiotic resistance, an effective vaccine is highly needed. extracellular products (ecps) have an essential role in phdp virulence, containing important antigens. however, the ecps' identity remain undisclosed. in our efforts to dissect their composition, we found that they contain high amounts of outer membrane vesicles (omvs). these particles are potent weapons for bacteria and are being explored in the field of vaccinology, since omvs present antigens in native conformations and are strongly immunogenic, without requiring adjuvants. this potential associated to the urgent need for an anti-phdp vaccine prompted us to isolate and characterize the omvs shed by phdp. methods: in order to harvest high amounts of pure phdp omvs, a reproducible optimized protocol was developed: the bacteria-free supernatant from a phdp overnight culture is concentrated, dialysed and ultracentrifuged to collect the omvs. results: analysis of the obtained omvs preparations by transmission electronic microscopy and dynamic light scattering indicate that the main population of vesicles has sizes around 30-50 nm. proteomic analysis of the vesicles revealed the presence of the apoptogenic ab toxin aip56 that is known to play a major role in phdp virulence, a putative pore-forming toxin, a putative adhesin/invasin and several outer membrane proteins (omps), including a 64kda omp, predicted to be involved in iron acquisition, and 5 other omps (18-34kda), with an ompa-like structure that may act as adhesins. moreover, preliminary in vivo studies suggest that some of those proteins may have important roles for virulence, since injection of knock-out strains in sea bass induced a decreased mortality comparing to the wt strains. summary/conclusion: our findings suggest that omvs are a promising vaccine candidate and we are currently studying their biological activities and determining the antigenic potential of the identified proteins. introduction: whole body exposure to high doses of ionizing radiation (ir) can potentially be lethal if radiation injury is not diagnosed and treated expeditiously. when considering a non-invasive approach for the identification of biomarkers of ir exposure, we and others have studied molecules in plasma, serum, saliva, and urine. however, these matrices can potentially have significant background noise, obscuring potential biomarkers of biological importance. extracellular vesicles (evs) are fast becoming a platform for biomarker discovery in radiation research as well as in other pathologies. however, no groups have investigated the use of metabolomics to analyse evs derived from urine in the context of ir exposure. furthermore, the dominant protocols for ev isolation from urine require a large (up to 30 ml) amount of starting volume, which may not be available for many studies. the aim of this study was to optimize ev isolation from rat urine and assess radiation-induced alterations in urine ev number and metabolic content. methods: as a proof of concept, we compared and optimized several ev isolation methods on small volumes of urine from male wag/rijcmcr rats exposed to 0 gy or 13 gy x-rays to the whole body except the hind leg. starting with either 500 µl or 1000 µl of urine, we isolated evs using ultracentrifugation (uc) with filtration, size exclusion chromatography (sec), and a proprietary bead-based isolation method developed by a 3rd party provider. ev samples were characterized using nanoparticle tracking analysis. metabolomics profiles were measured using lc-qtof-ms. results: we found that sec resulted in the highest yield of evs from as little as 500 µl of urine, while uc was the poorest performing. lc-qtof-ms analysis revealed that sec and uc had the most consistent identification of features, whereas the bead-based method contained artefacts likely as a result of the extraction method. we next used sec to isolate evs from a larger cohort of rats exposed to ir and analysed with ms. ev metabolic content will eb related to differences in survival and organ function between sham and irradiated groups. summary/conclusion: we conclude that sec is the preferred method for isolating evs from small volumes of urine for broad-based mass spectrometric analysis, and that the ev metabolome may be a sensitive and specific early indicator of radiation injury. introduction: there is growing evidence that contents (including rna and proteins) of exosomes may serve as biomarkers for early diagnosis and prognostic prediction of cancers. here we aim to identify potential protein markers for oesophageal cancer. methods: using our newly developed label-free exosome automated preparation system (leaps), exosomes were isolated from 20 ml culture medium of various oesophageal cancer cells with different differentiation profiles and different sources of metastasis. exosomes from 20 µl plasma of cancer patients at different clinical stages or with/without relapse and healthy controls were also prepared by leaps. matrix-assisted laser desorption ionization time-of-flight mass spectrometry (maldi-tof ms) was employed to directly analyse exosomes. protein identities of exosomal fingerprint peaks were tentatively assigned by correlation with top-down and bottom-up proteomics. results: start from 20 ml culture medium or 20 µl plasma, high-quality exosomes rapidly isolated by leaps are sufficient for maldi-tof mass spectrometry. it seemed that poorly differentiated cells showed more exosome release. maldi-tof ms fingerprints of exosomes in cells is cell line specific. ms profiles from poorly differentiated cells showed more peaks than that from highly differentiated cells. fingerprints also allowed classification of cancer cell lines through software mathematical analysis. we identified different numbers of significantly differentially expressed peaks in exosomes of various cancer cells. fingerprints of exosomes derived from the poorly differentiated cells showed more elevated peaks. top four peaks (2,427 m/z, 3,596 m/z, 4,458 m/z, 4 ,537 m/z) were commonly down-regulated in exosomes of most cancer cells. top four protein peaks (2,268 m/z, 5,713 m/z, 6,255 m/z, 6,337 m/z) that might be correlated to the differentiation profile of cancer cells were also identified. maldi-tof ms detection of exosomes in the plasma and clarifying identities of potential biomarker peaks will be done in the future. summary/conclusion: the combination of leaps and maldi-tof mass spectrometry provides a fast and high-throughput tool for exosomal marker discovery. potential biomarker identified in exosomes derived from oesophageal cancer cells or from plasma of cancer patients by this tool might be useful in cancer diagnosis and prognosis. fraction-based proteomic profiling of serum extracellular vesicles derived from cervical cancer patients introduction: current evidence indicates that extracellular vesicles (evs) can release from most of cell types and affect adjacent or distant cells by circulating in all bodily fluids. proteomic analysis of evs from clinical samples is complicated by the low abundance of ev proteins relative to highly abundant circulating proteins. size exclusion chromatography (sec) has been overcome as a method to deplete protein contaminants and enrich evs. methods: we collected serum of healthy women and cervical cancer patients with stage i-iii and then counted concentration and size distribution of the evs using nanoparticle tracking analysis (nta). differential ultracentrifugation combined with sec was used to isolate and purify evs from contaminant proteins. isolated evs were investigated their characteristic based on morphology using transmission electron microscope (tem) and on expression of cd63, cd81, cd9 protein markers using western blot analysis. fraction no.8-10 of isolated evs in among sample groups were profiled by nano-liquid chromatography tandem mass spectrometry (nanolc-ms/ms) analysis. results: nta shows that the concentration of evs is increased in patients compared with healthy women. proteome profiles of evs isolated by sec were compared in each fraction. moreover, we detected molecular evidence for fraction-specific molecular pathways in connection with cancer progression and complied a set of protein signatures that closely reflect the associated clinical pathophysiology. summary/conclusion: these unique features in each fraction among sample groups would be the informative considering in order to select for further analysis as in vitro. introduction: recently, diagnostic biomarkers from exosomes by proteomic analysis have been reported, but it is required to optimize the isolation protocol to screen out more effective biomarkers. for serum-originated exosomes, it has been also reported to isolate them selectively, however, it is observed that a different method resulted in different protein profiles in 2-d gel electrophoresis. methods: we isolated exosomes by two discrete methods, using ultracentrifugation and magnetic separation. before ultracentrifugation and magnetic separation, precipitation using polymer materials was perforemd. the isolation of exosomes by these two methods followed by comparison of their size, total vesicle number, morphology, and protein markers. to identify protein biomarkers, proteomic analyis using 2-d gel electrophoresis was performed. results: both methods induced enrichment of exosome-specific proteins, but protein profiles in each exosome fraction was totally different. the protein profiles showd that the magnetic seperation following a polymer-based precipitation step was more efficient to screen out candidate biomarkers, which showed nearly 200 protein profiles originated from exosomes. summary/conclusion: in our study, magnetic separation of exosomes from serum fraction was optimized for 2-d gel electrophoresis to observe identifiable biomarkers. an extracellular small rna-seq data processing pipeline optimized for high-performance computing chenghao zhu and angela zivkovic department of nutrition, uc davis, davis, usa introduction: a variety of rna species is found in extracellular biofluids such as blood, bile, and urine, carried by extracellular particles including extracellular vesicles (evs) and lipoproteins (e.g high density lipoproteins (hdls)). the extracellular rna (exrna) carried by evs and hdls is of great interest for two reasons: 1) the exrna within different carriers could be diagnostic of the state of the tissues from which the particles originate, and 2) exrna has been shown to affect gene expression in target cells. although the origin and functions of exrnas remain largely unknown, there is growing interest in exrna research for the development of diagnostics and new therapeutic targets. small rna sequencing is widely used to estimate the abundance of exrnas in biofluid samples. here we present a data processing pipeline for extracellular small rna sequencing. sequencing data are pre-processed through quality control, and then aligned to the endogenous genome to obtain the gene counts for various rna biotypes, including microrna, trna, rrna, piwi-interacting rna, long non-coding rna (lncrna) and protein coding rna. it also aligns sequencing reads to exogenous databases, including the ribosomal rna sequence database silva, and all sequenced bacteria genomes available on ensembl, to estimate the abundance of exogenous genes. results: we analysed a publicly available small rna-seq dataset of hdl from three systemic lupus erythematosus (sle) patients and three healthy controls using this pipeline. the mirna hsd-mir-93, lncrna al137186.2 and ac108050.1 were elevated in sle patients compared to controls. exogenous rna reads mapped to bacteroidetes were also elevated in sle patients. summary/conclusion: our pipeline is able to process exrna sequencing data and estimate the abundance of major exrna species, as well as exogenous rna taxonomy. the pipeline is optimized for the job scheduler slurm, and can therefore utilize the full computational power of high-performance computers. the pipeline is publicly available on github (www.github. com/zhuchcn/excernapipeline). introduction: ibd is a chronic hyperinflammatory disorder that severely compromises the intestines. the aetiology of ibd is poorly understood. however, it has been associated with a dysregulation of the immune system and gut microbiota and with genetic and environmental factors. cumulative evidence indicates that evs play an essential role in modulating immune responses. recent research suggests that evs derived from dendritic cells, saliva and intestinal epithelial cells may be involved in the progression of ibd inflammation. however, little is known about the contribution of immune cells-derived evs with this pathology. the goal of this study is to shed light on the contribution of pbmc-derived evs on ibd pathogenesis. here we characterized and compared the composition of evs derived from pbmcs of 3 ibd patients and 1 healthy control. evs were isolated by differential centrifugations from the supernatant of pbmc activated with cd3-cd28 beads for 6 days in serum-free media. size and concentration were analysed using a nano sight 300 instrument, while the presence of known evs markers (cd63, cd81, hsp70) was analysed by immunoblotting. whole evs proteome was performed by ms/ms and functional-enrichment analysis was done using funrich with uniprot database. results: proteomics analyses identified a total of 1299 proteins in the four groups. of those, 673 (51.8%) were present in both the ibd patients and control. this group of protein was composed of several ras-related proteins, eukaryotic initiation factors, granzyme, cd9, tubulin, and serpins among others. patients' evs shared 46 proteins in common such as proteasome subunit beta type-10, t cell receptor beta, and the amine oxidase containing copper 3. interestingly, each patient sample had a unique group of proteins. among these are myeloperoxidase, neutrophil elastase, proteasome subunit alpha type-3, and signalling lymphocytic activation molecule (slamf1). summary/conclusion: these preliminary studies show that the ev composition from pbmcs of ibd patients is specific and differs from a healthy control. this exclusive composition has the potential to be used as a biomarker for diagnostics and progression of the disease, and it could also provide new insights into our understanding of the cellular pathways involved in the pathogenesis of ibd. the studies were performed with corresponding irb approvals. proteomic analysis of exosomes isolated using precipitation and columnbased approaches introduction: exosomes are a subtype of small extracellular vesicles (evs) involved in various physiological and pathological processes with huge potential as biomarker resources or as therapeutic tools. although several exosome isolation approaches are available, complementary studies focusing on optimizing the methods for human bloodderived exosomes isolation and method-specific comparative exosomal proteomic profiles will be of clinical value. methods: blood-derived evs were isolated through precipitation-and column-based methods and characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. serumderived exosomal proteomes were analysed by mass spectrometry (ms). the resulting proteomes were then overlapped with the proteomes obtained from exosome-related databases, to determine the % of similar content. in addition, bioinformatic analysis, including gene ontology (go) was carried out. results: both methodologies tested isolated particles with the expected morphology and size range, although the column-based method isolated a higher number of particles. about 75% of the exosomal proteins identified through ms overlapped with the proteomes extracted from the databases. go terms were similar for the proteomes isolated from the column-and precipitationbased methodologies. the top 3 go terms identified for molecular function were ion binding, peptidase activity and enzyme regulator activity and for biological process were immune system process, transport and response to stress. further, partial least square analysis revealed a clear segregation of proteomes obtained by the distint methodologies and complementary statistical analysis revealed the proteins differently expressed. summary/conclusion: no major differences were found in the top 3 biological processes and molecular function based on go analysis. nonetheless, the two approaches result in different evs yields and significant proteome differences were identified. characterization of distinct methods for blood-derived exosomes isolation can be useful in the context of evs potential in disease diagnostics/therapeutics. introduction: we and others are developing biomarkers for neurodegenerative diseases using neuronalenriched evs immunocaptured from a suspension of total plasma evs. here we assess how the isolation method for total evs affects the yield, purity and enrichment of neuronal evs. methods: for n = 5 subjects, total evs were isolated by ev precipitation solution (exq), ev precipitation solution plus bipartite resin columns (exu) and size exclusion chromatography (qev) from 0.5, 0.5 and 2.7 ml plasma, respectively. then, neuronal-enriched evs were immunoprecipitated using anti-l1cam antibody. in total and l1cam evs, we measured particle concentration by nanoparticle tracking analysis, protein concentration, and novel multiplex electrochemiluminescence immunoassays for tetraspanins cd81, cd63 and cd9 on intact evs. results: for total evs, yield followed the order of exq > qev > exu, assessed by particle (p < 0.0001) and protein concentrations (p < 0.001). l1cam evs immunocaptured after exq showed 16-fold higher particle (p < 0.001) and fivefold higher protein (p < 0.01) concentrations compared to l1cam evs after exu, and 41-fold higher particle (p < 0.001) and 11-fold higher protein (p < 0.001) concentration compared to l1cam evs after qev. l1cam evs after ev precipitation (exq) showed 48, 35 and 30-fold higher cd81, cd63, and cd9 concentrations (p < 0.001) compared to l1cam evs after exu, and 59, 43 and 36-fold higher cd81, cd63, and cd9 concentrations (p < 0.001) compared to l1cam evs after qev. l1cam evs following 3 different methods had equal purity assessed by ratios of particle/protein concentrations (p = ns), and tetraspanin/particle concentrations (p = ns). summary/conclusion: l1cam ev immunocapture preceded by exq exceeded the yield of immunocapture preceded by exu or qev. recovered l1cam evs showed equal purity by particle/protein and tetraspanin/particle metrics. neuronal enrichment results will be available by the time of isev. immunoprecipitation following exq, often considered impure, purifies final isolates as effectively as more onerous methods typically considered purer. balancing sensitivity, purity and scalability is essential for implementation of blood biomarkers in the clinical setting and may be achieved by combining techniques. funding: this research was supported in part by the intramural research program of the nih, national institute on aging. characterisation of breath exosomes: towards non-invasive diagnosis deanna ayupova a , renee goreham b and paul teesdale-spittle c a school of chemical and physcial sciences, victoria univeristy of wellington, wellington, new zealand; b university of newcastle, newcastle, australia; c school of biological sciences, victoria univeristy of wellington, wellington, new zealand introduction: breath-derived exosomes present new potential for non-invasive diagnosis of lung cancer. however, breath-derived exosomes have not been well characterized and methodology for their purification has not been optimised. in order to exploit their potential for diagnosis, it is first necessary to develop methods that reproducibly provide high quality pure exosomes from breath. in this study, we optimise methods for their isolation and characterise them in comparison to exosomes derived from cell culture models. methods: in order to characterize exosomes from exhaled breath condensate (ebc) it was first necessary to optimize methods for isolation of pure, intact, and high quality exosomes. to this end, isolation methods were optimised on cell-derived exosomes and then applied to ebc, yielding high quality exosomes from size exclusion chromatography (sec). ebc exosomes were compared with those from a549 and wi-cells using dls, tem, and cryo-sem. an immunoblotting-grid technique was used to validate the presence of exosome-specific markers cd63 and cd81. protein content of exosomes were quantified and compared. results: sec-based isolation was more effective at isolation of pure and intact exosomes than ultracentrifugation, with the highest purity exosomes obtained in the middle fractions of the exosome-containing eluate. exosomes from ebc had a size range (45-100 nm), protein content (20-40 ug/ml) and molecular markers typical of cell-derived exosomes. summary/conclusion: breath-derived exosomes isolated through size exclusion chromatography are sufficiently pure for diagnostic purposes and are phenotypically similar to exosomes derived from other sources. we foresee their use in non-invasive diagnostics for lung cancer as an important future application. ligand-based exosome affinity purification (leap) is a rapid and reproducible method for the enrichment of functional evs introduction: platelet-derived extracellular vesicles (pevs) represent the next generation of therapeutic biologics as they enable a more refined and targeted approach when compared to crude blood derivatives currently used for treating diseases such as cancer, thrombocytopenia and chronic wounds. however, development of an ev-based therapeutic is hindered by the lack of a scalable, validated and reproducible purification process. in this study, pevs were isolated from activated platelet concentrates and purified using exopharm's ligand-based exosome affinity purification (leap) technology to produce a functionally active ev therapeutic. methods: platelet concentrates (n = 20) were obtained from the australian red cross blood service and were activated by exopharm's proprietary process. activation was verified by measuring cd62p using flow cytometry. the resulting platelet releasate (500 ml) was subjected to leap purification to isolate pevs. for characterization, protein concentration was determined by a bicinchoninic acid assay, microfluidic resistive pulse sensing (mrps) was used to perform a particle count and transmission electron microscopy (tem) enabled visualization of ev morphology. key ev markers were detected using mass spectrometry (ms) and western blots. to confirm biological activity, human dermal fibroblasts were subjected to serum starvation for 16 hours before treatment with pevs (15 µg/ml). cell growth was recorded by the real-time xcelligence system and differences in proliferation were statistically analysed using a one-way anova. results: mrps and tem both revealed isolated pevs to be 100-300 nm in size. the final product was positive for platelet markers (cd41, cd62p) and key ev markers (tsg101, alix, cd63). treatment with purified pevs significantly increased proliferation in serumstarved fibroblasts over 48 hours. summary/conclusion: exopharm's leap technology is a rapid and reproducible purification process which produces pevs that adhere to misev 2018 guidelines and are functionally active. funding: all funding was through exopharm ltd (asx:ex1) a novel but simple method to obtain purified exosomes by one-step ultracentrifugation introduction: exosomes are extracellular vesicles (evs) that are derived from endosome membrane. they are usually 50-150 nm in diameter, actively secreted in most living cells. originally, exosomes were thought to act as cellular garbage disposals. recent studies showed that exosomes not only can serve as biomarkers for diagnosis, but also can be used as an ideal delivery vehicle for drugs in therapeutics. exosomes are natural carrier for mrna, mirna, sirna, protein, dna and peptide for long distance intercellular communication. isolation of exosomes is challenging due to their small size and heterogeneity. traditional differential ultracentrifugation method is still the gold standard for exosome purification. to further explore the potentials of exosomes being as the therapeutic delivery vehicle or diagnostic reagent, it is an essential step to purify them in high quality at high yield. methods: here, we report a novel method to obtain intact shape, high-quality and high purity exosomes with one-step ultracentrifugation by using "exojuice". results: data of nanoparticle tracking analysis (nta) and western blotting showed "exojuice" can yield exosomes with a simpler method to obtain higher purity exosomes in comparison to previous method of cushion ultracentrifugation using optiprep. summary/conclusion: our method can be used to purify exosomes from cell culture medium, serum, urine, saliva, and other biofluids. a straightforward device to extract apoplastic fluid from succulent fruits for higher purity of extracellular vesicles introduction: edible plants are emerging as a sustainable source for extracellular vesicle (ev)-based drug delivery vehicles. however, current isolation methods (e.g. grinding or squeezing) may cause destruction of plants' biostructures, and in turn leads to unwanted effects in downstream applications and complicates the study of nanovesiclescell. therefore, we designed a simple device that allows the extraction of apoplastic fluid (af) from succulent fruits, facilitating ev isolation as well as effective downstream applications. methods: an inner filter tube was designed to extract af with a determined membrane pore size. af was collected by low-speed centrifugation method and then filtered to eliminate the impurities from the cytoplasm and damaged cells. minced juice (mj) was homogenized by a blender and then centrifuged to remove large fragments. subsequently, the differential centrifugation method was employed to extract evs from af and mj. fourier-transform infrared spectroscopy (ftir), nanoparticle tracking analysis (nta), and transmission electron microscopy (tem) were performed to discriminate af, mj and their evs. results: the "spectroscopic" protein-to-lipid (p/l) ratio of af (1.23 ± 0.03) is significantly lower than that in mj (1.27 ± 0.01), showing the higher lipid contents in af, which may result from the loss of lipids in mj obtained from grinding or juicing methods. similarly, ftir showed the difference in p/l ratio between af and its evs (1.23 ± 0.03 and 1.29 ± 0.02, respectively). nta showed the sharper peak and smaller vesicle size in the following order: mj (173.0 ± 21.3 nm), af (154 ± 11.0 nm), af-derived evs collected at 40,000 × g and 10,000 × g (108.5 ± 2.4 nm and 95.7 ± 3.1 nm, respectively). furthermore, tem study indicated that the collected evs exhibited a typical lipid bilayer of extracellular nanovesicles. summary/conclusion: by using a reusable filter device, we successfully isolated af from succulent fruits, paving the way to collect plant evs without an interference of significant biodestruction or damaged cells, hence improving the purity of evs and facilitating downstream applications. moreover, this method is straightforward, reproductive, and can be potentially used in a large-scale production. method to simultaneously capture multiple classes of intact extracellular rna carriers including extracellular vesicles and lipoprotein particles introduction: extracellular particles including extracellular vesicles (evs), lipoproteins, and free proteins are carriers of extracellular rna (exrna), which has been shown to regulate cellular function. because these particles have different physiological origins, they have different rna signatures, so the first step to understanding the biology of exrna is to isolate individual particle fractions with high purity and efficiency. current methods for isolating evs are optimized for increased yields and purity of ev fractions but typically require multiple millilitres of starting plasma and do not capture the other exrna carrier particle types. methods that can capture evs from low starting plasma volumes and can also capture other exrna carriers simultaneously are needed for analysing samples from previously conducted large cohort studies, biorepositories, and in populations where sample volume is limiting. methods: we have developed a method adapted from lipoprotein isolation that requires only 500 µl of starting plasma, and uses brief ultracentrifugation (uc) followed by fast protein liquid chromatography (fplc) to capture 6 classes of purified exrna carriers including evs, ldl, hdl, lipidated albumin, proteins, and vldl/chylomicrons. we have validated successful capture of evs by microfluidic resistive pulse sensing (mrps, spectradyne), transmission electron microscopy (tem), and single particle interferometric reflectance imaging system (sp-iris; exoview) with optional fluorescence. results: we have observed 1.02 × 1010 particles per ml from a 1 ml fplc fraction of evs measured from 25,000 events by mrps, confirming that evs are being captured by this method. there were also 1.26 × 1010 particles/ml and 2.70 × 1010 particles/ml in the two subsequent 1 ml fractions that are known to contain lipoprotein particles, though these were measured from 1,500 events each. by tem we confirmed these observations that evs are eluting before lipoprotein particles with some evs eluting later in fractions containing lipoproteins. summary/conclusion: these results confirm the efficacy of the method in isolating multiple exrna carrier fractions simultaneously from a single 500ul plasma sample, making it amenable for the analysis of exrna in samples from large cohort studies, biorepositories, and vulnerable populations such as the elderly and young children. funding: nih/nia r01ag062240; nih ug3 ca241694-01 optimizing the isolation of placental mesenchymal stromal cell-derived extracellular vesicles in a 3d bioreactor system leora goldbloom-helzner a and aijun wang baaaaa a uc davis, davis, usa; b uc davis medical center, sacramento, usa introduction: extracellular vesicles (evs) derived from placental mesenchymal stromal cells (pmscs) have the potential to provide neuroprotection at sites of injury. however, a rate limiting step in ev research is the low yield, high technical time, and high cost of current isolation procedures. to address this inefficiency, we cultured pmscs in a unique bioreactor system to increase the absolute yield of evs per ml of media and per cell. future studies will determine if this system can improve pmsc ev yield without altering the demonstrated neuroprotective properties of pmsc-evs. methods: pmscs were cultured in the bioreactor for ten weeks. ev-conditioned media was collected weekly and evs were isolated through differential centrifugation. nanoparticle tracking analysis (nta) measured ev size and concentration. western blots tested for normal ev markers (cd9, cd63, and cd81, calnexin(-)) and enzyme-linked immunosorbent assays (elisa) measured levels of characteristic growth factors in conditioned media including vascular endothelial growth factor (vegf), brain-derived neurotrophic factor (bdnf), and hepatocyte growth factor (hgf). results: evs remained consistent until week eight, after which a decrease in both ev size and concentration was seen. western blots revealed normal positive expressions of cd9, cd63, and cd81 and negative expressions of calnexin. levels of vegf, bndf, and hgf were comparable after 10 weeks. cost analysis revealed an overall increase in ev yield for shorter labour time and lower material cost. summary/conclusion: this initial study uses a bioreactor system for a unique source of cells and has brought us closer to optimizing pmsc ev isolation protocols for increased yield, lower cost and time commitment, and maintained sample purity. preliminary data suggests the ev phenotype and cell secretome are consistent with those present in current culture settings. future experiments will assess the preserved neuroprotective properties of the pmsc evs. a novel method for isolating extracellular vesicles from cell culture media and plasma using polyethylenimine introduction: due to their ability to transport dna, rna, and protein cargoes between cells, extracellular vesicles (evs) are becoming popular for biomarker discovery as well as for therapeutic delivery. here we describe the development of a novel precipitation method for the isolation of evs from cell culture media and plasma that is based on polyethylenimine (pei), an inexpensive, water-soluble, and biocompatible cationic polymer. pei is a group of hydrophilic cationic polymers that are synthesized as either linear or branched forms of varying molecular masses (1,000 to 750,000 da) and are widely used in the biomedical field as a coating and transfection agent. methods: linear and branched pei of varying molecular weights (mw) were tested for their ability to precipitate evs from either conditioned culture media (ccm) or human plasma. isolated evs were characterized by western blotting and nanoparticle tracking analysis (nta). the small rna profile of evs isolated using pei from human plasma was analysed by ngs and ev-specific mirnas were confirmed by digital droplet pcr (ddpcr). mass spectrometry (ms) was used to analyse the proteome of pei-captured evs from plasma. hek293 cells producing gfp+ evs were used to optimize conditions for release of evs from both linear and branched pei by fluorescent spectrophotometry and flow cytometry measure-ments. results: linear and branched pei were both able to precipitate evs as determined by western blotting for ev protein markers; however, branched pei with mw > 10,000 da and linear pei with mw > 25,000 da were more efficient for ev precipitation than lower mw forms. despite its known ability to bind nucleic acids pei was unable to capture cell-free dna from plasma, although rna and in particular ev-associated mirnas such as mir-142-3p were recovered. ms revealed that pei enriches extracellular exosome proteins from plasma. evs captured from ccm by pei could be released from the complex using heparin or high salt conditions. summary/conclusion: pei has an unexpected preference for associating with evs compared to nucleic acids in complex biological samples and has a hitherto unrecognized application for ev precipitation. introduction: there is ongoing debate about which is the most appropriate method for isolation of evs, with most labs using some combination of differential ultracentrifugation (uc), size-exclusion chromatography (sec), and/or density gradient ultracentrifugation (dg). here we applied a surface-enhanced raman spectroscopy (sers) analysis platform to compare chemical composition of the isolate from each method against lipoprotein standards to assess the relative purity of the ev preps. methods: 2-3 ml of plasma was separated from whole blood collected from head and neck cancer patients. each sample was split into 3 batches and evs were isolated by either uc, sec, or dg. following isolation, samples were incubated on commercial sers substrates and raman spectra were collected. lipoprotein standards were purchased and also measured for comparison. using principle component analysis (pca), spectra were analysed for chemical variability. results: sers analysis of sec, uc, and dg isolated evs were chemically distinguishable using simple pca. the chemical changes could in large part be attributed to fitting the differences in spectra to lipoprotein standards. we found that uc isolated populations clustered with the high-density lipoproteins (hdl), sec populations with the low-and very low-density lipoproteins (ldl, vldl), and dg populations were more variable, but mainly clustered together with the highdensity-lipoproteins (hdl). summary/conclusion: this set of experiments matches our expectation that various lipoprotein would contaminate ev preps according to their relative size and density distributions. no single isolation method could separate pure ev samples. this study also illustrates the utility of label-free sers analysis for rapid chemical characterization of evs. bioreactors: lessons to develop an extracellular vesicle factory vanessa chang, priscila dauros-singorenko, lawrence w. chamley, colin l. the university of auckland, auckland, new zealand introduction: high density mammalian cell culture systems (bioreactors) provide valuable advantage for large scale production of secreted products such as extracellular vesicles (ev). however, optimisation of design selection, handling and operational costs can be quite challenging. here we provide our experience with a celline bioreactor system. methods: cultures of 6 adherent cell lines were established in celline ad 1000 bioreactors and propagated for up to 19 weeks. media was changed twice weekly and cells shed into serum-free conditioned medium were counted and assessed for viability. nanoevs were isolated by sequential centrifugation (2000 g -10,000 g -100,000 g) and size exclusion chromatography (sec). nanoevs were characterised in their protein (bca) and particle (nanoparticle tracking analysis) amount, ev markers (western blotting) and morphology (transmission electron microscopy, tem). results: the viability of shed cells varied between cell lines and through time, suggesting a changing dynamic during reactor establishment and continuous growth phases, that was specific to each cell line. hdfa, bt20 and bt474 consistently shed mainly dead cells (60-100%), as opposed to mcf7 and mda-mb-231 which predominantly shed live cells. sec fractionation of nanoevs identified a dominate ev-rich peak and significant quantities of smaller proteins, highlighting the need for further purification. nanoev yields from each 3-4 day culture averaged 2-7 × 1010 particles, representative of yields obtained from cells grown in 8 to 28 conventional t175 tissue culture flasks. ev markers and tem confirmed the protein profiles and morphology of evs obtained from bioreactors. summary/conclusion: high density bioreactor cultures offer a physiologically relevant, cost and space efficient approach to produce significant amounts of evs, providing sufficient material for numerous experimental uses. in our hands, with careful twice weekly management, they can be propagated for up to 19 weeks without significant changes to the evs. introduction: extracellular vesicles (evs) have potential applications for clinical theranostics. ultracentri-fugation is most commonly adopted to the evs isolation, which is recommended as a gold standard method. however, ultracentrifugation is time-consuming and expensive equipment requirement, resulting in the coisolation of contaminants such as protein aggregates. therefore, our aim is to develop a rapid and efficient platform to isolate heterogonous evs based on the insertion of lipid molecules into the evs membrane to avoid co-isolation of non-membranous protein particles. methods: herein, a defected nanoscale functional metal organic framework (mof) was constructed as an efficient platform for evs isolation. typically, one single-stranded dna was designed and modified with a phosphate group at the 5ʹ-end and cholesterol at the 3ʹ-end to form a capture dna named phosphate−dna−cholesterol (pdc). the phosphate group forms a strong covalent bond with the designed defeated site of zr (iv) in mof uio-66-nh2 and the cholesterol inserts into the phospholipid bilayer to capture evs without non-membranous particles contamination. the formed mof−phosphate−dna −cholesterol−evs (mof@pdc@evs) system was further treated with dnase i for dna hydrolysis to give high pure evs. results: a rapid and efficient isolation platform of evs based on a defected mof functionalized with phosphate-dna-cholesterol (mof@pdc) has been constructed successfully. compared with ultracentrifugation, mof@pdc platform promises to isolate size heterogeneous evs i) without non-membranous particles contamination, maintaining evs intact membrane structure, protein components, and biological functions; ii) with the ability to capture evs with 78% isolation efficiency; iii) makes evs isolation process simple and fast, which could be finished in 40 minutes without requirement of the expensive equipment. summary/conclusion: in conclusion, this rapid and efficient platform is suitable for isolation evs from biological fluid for downstream protein analysis. this work opens a new perspective in mof-based separation researches and may shed light on further studies towards evs isolation. introduction: incorporation of pharmacologically active molecules on the surface or the lumen of extracellular vesicles (evs) is an important strategy for maximizing the therapeutic potential of evs. genetic engineering of producer cells by introducing dna through random or site-specific integration are promising strategies for creating engineered evs. longterm stability with consistent transgene expression in the ev producer cells and therapeutic potency of resulting engineered evs are crucial for biomanufacturing. we present a comprehensive study to investigate stability of transgene expression and potency of two potential therapeutic engineered evs derived from stably selected pools transfected by either random integration (ri) or site-specific integration (ssi). methods: producer cells were engineered to make evs displaying interleukin 12 (il12) or interferon gamma (ifng) by ri or nuclease-mediated ssi into aavs1 locus. following puromycin (puro) selection, longterm cellular stability and transgene expression without selective pressure was investigated. evs were generated from stable cell pools at 0, 1, and 2 months post-thaw and purified by density gradient ultracentrifugation. purified evs were biochemically characterized by nta, bca, western blot, and cholesterol quantitation. transgene expression and biological activity of evs displaying il12 and ifng were assessed by alphalisa and in vitro reporter assays. results: transfection by ssi resulted in faster recovery in puro selection compared to ri. all stable cell pools, regardless of integration method, resulted in comparable cell culture performance, ev yield, and lipid and protein content at all time points tested. the engineered evs also demonstrated long-term stability of il12 and ifng transgene expression and in vitro activity from both integration strategies. summary/conclusion: both methods for generating stable cell lines were comparable in terms of cell stability, transgene expression, ev titre and potency, with ssi having the advantage of speed, allowing for more rapid iteration cycle times. thus, both methods are suitable for the precision engineering of therapeutic evs. this work demonstrates feasibility to manufacture therapeutic engineered evs from stable cells from either integration strategy for clinical development. transport of outer membrane vesicles as a model therapeutic delivery system in pathogenic and commensal bacteria introduction: outer membrane vesicles (omvs) in gram-negative bacteria have been shown to be important carriers of biomolecules, including toxins and other virulence factors, peptidoglycan, and nucleic acids. it has been shown that omvs play an important role in the delivery of these biomolecules to host cells and bacterial cells. while many thorough studies have explored omv delivery to host cells, few studies have explored the mechanisms of delivery of omvs to bacterial cells. our goal was to study the delivery of omvs to other bacterial cells. specifically, we were studying the oral pathogen aggregatibacter actinomycetemcomitans (a.a.), a gram-negative organism associated with localized aggressive periodontitis, to study the process by which vesicles from this organism communicate with other bacterial cells. overall, we want to understand the roles specific surface components of omvs play in the transport of these omvs to other bacterial cells. methods: we studied omvs from two strains of a.a.: jp2, a highly pathogenic strain, and 33384, a natural commensal strain. af488-labelled omvs were incubated with fresh bacterial cultures. association of the omvs with the bacterial cells was quantified using flow cytometry. to examine the role of surface-associated dna in this process, dna was digested with dnase, and the amount of surface-bound dna was quantified with the membrane impermeable dna stain, toto-1. results: using flow cytometry, we observed jp2 omvs were delivered to 33,384 cells, and at a lesser amount to jp2 cells. alternatively, 33,384 omvs associated readily with jp2 cells, more than to 33,384 cells. this suggests that the delivery of omvs to bacterial cells may be a targeted delivery mechanism. furthermore, we hypothesized surface-associated dna may play a role in this interaction. we next digested the surface-associated dna on the omvs with dnase, and observed a decrease in association between the omvs and bacterial cells. this supports our hypothesis that dna on the surface of the omvs plays a role in association. current experiments are investigating this interaction in more detail. summary/conclusion: we have demonstrated that omvs are selectively delivered to bacterial cells, and surface-associated dna plays a role in this process. we propose to investigate this process to further understand omvs delivery to bacterial cells. funding: r03de025275 & r21de027769. utilizing a gaucher's disease cell line for the evaluation of a novel exosome-based replacement therapy annie k. brown a , jiayi zhang b , brendan lawler b and biao lu b a santa clara university, san jose, usa; b santa clara university, santa clara, usa introduction: engineered nano-scale exosomes have great potential as new and targeted delivery vehicles for the treatment of gaucher's disease, the most common lysosomal storage disease. recently, we have reported the design, production, and isolation of exosomes loaded with lysosomal β-glucocerebrosidase (gba). people suffering from gaucher's disease do not have functional gba, which results in toxic build-up of undegraded substrates within the cell. methods: to evaluate the efficacy of this exosomebased therapy, a human gaucher's disease model is required. here, we have utilized near-haploid human cells (hap1) modified via crispr-cas9 to model gaucher's disease in vitro. these cells contain a 479bp insertion in the 6th exon of the gba gene, resulting in non-functional gba. pcr, enzyme activity assays, and flow cytometry have been employed to confirm the diseased genotype and phenotype. results: characterization of gba-knock out cells shows a total loss of gba enzyme activity. further characterization demonstrates a normal growth rate but an increased number of lysosomes, indicating a diseased phenotype. summary/conclusion: the utilization of a human gba-knock out cell line will enable the evaluation of the efficacy of our engineered exosomes. disease models will be an important resource for the evaluation of new biologic therapeutics, including exosomes. funding: we would like to acknowledge the santa clara university school of engineering for their support. thrxosomes: a novel exosomes based theranostic for lung cancer introduction: chemotherapy is the first-line of treatment for lung cancer. however, inefficient bio-distribution and reduced accumulation of drugs in the tumour results in treatment failure. therefore, improved drug delivery and diagnostic systems are warranted. herein, we propose a novel theranostic system "thrxosomes" where exosomes are loaded with super paramagnetic iron nanoparticles (spions) conjugated to an anticancer drug via a phresponsive linker for controlled release. we hypothesize that thrxosomes will exert profound anticancer tumour activity that can be concurrently be monitored by magnetic resonance imaging (mri). methods: thrxosomes were produced by combining normal human lung fibroblast (mrc9) cell-derived exosomes with spions conjugated to and anti-cancer drug (chemodrug or mirna) via a ph cleavable linker. the physical and biological properties of thrxosomes were determined using transmission electronic microscopy (tem), nanotracker-analysis (nta), inductively coupled plasma mass spectrometry (icpms), western blotting, cell viability, and mri. results: exosomes used in preparing thrxosomes were 130 nm in size with a typical lipid bilayer structure, and were positive for cd63, cd81, flotillin and negative for annexin a1 confirming presence and purity of exosomes. charge analysis, tem, and icmps data showed successful loading of spion-drug conjugate. biological studies showed selective and enhanced drug release under acidic condition (ph 5.5) compared to drug release at ph 7.2. cell uptake and viability studies demonstrated increased uptake and killing of thrxosome-treated human a549 lung cancer cells compared to mrc-9 cells. in vivo studies demonstrated accumulation and detection of spions by mri in in-situ tumours of a549 tumour-bearing mice. summary/conclusion: our study demonstrates thrxosomes will produce profound anticancer activity in lung cancer that is measurable by mri. exosome-modified nanoparticles as an alternative delivery system for small rnas in cancer therapy petro zhupanyn a , alexander ewe b , thomas büch c and achim aigner a a independent division for clinical pharmacology at rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany; b dr., leipzig, germany; c rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany introduction: gene knockdown by rna interference (rnai) is an alternative, non-invasive method for inhibiting proliferation or promoting apoptosis in tumour cells. this technique allows the specific targeting of key signalling proteins or mutated genes. most of the available transfection compounds suffer from rather profound cytotoxicity in vitro. the aim of our study was to establish a novel targeted small nucleic acid delivery system to the cells, with good cellular biocompatibility and applicability for in vivo studies. for this aim, we used native, cell own vesicles-exosomes. since exosomes are known to transport peptides and different rnas between cells and tissues, these unique, small extracellular vesicles (ev) may also be useful as transport vehicles for therapeutic sirna. methods: as detected by multiple cell surface protein expression analysis, exosomes carry specific surface expression markers, allowing the cellular uptake by the most of tissues. we established an ev purification protocol from tumour cell culture supernatants and a strategy for the efficient ev loading with our test sirnas or antimirs. here we used the combination of polyethylenimine (pei)-complexation of the rnas with ultrasound treatment for their loading into the evs. our ev-modified, ultrasound-treated nanoparticles were tested in vitro by measuring knockdown efficacies in luciferase reporter cell lines or by rt-qpcr gene expression analysis. results: more efficient cellular sirna uptake was observed upon ev-modification of our pei/rna nanoparticles, accompanied by efficient inhibition of gene expression. biological efficacies were retained also after storage for several days at room temperature. the monitoring of the ev-based particles by facs revealed a different time resolution of cellular uptake and nucleic acid release compared to the classically formulated peinanoparticles. in an in vivo therapy study in tumour xenograft-bearing mice, high biocompatibility, significant biological knock-down and tumour inhibition were observed after injection of anti-survivin sirnas formulated in our ecv-modified pei nanoparticles. summary/conclusion: our data demonstrate the usability of ecv-modified nanoparticles as efficient delivery system for small rnas in cancer therapy. microglial extracellular vesicles as therapeutic vector for neuroinflammation giulia marostica a , annamaria finardi b and roberto furlan a a san raffaele scientific institute, milan, italy; b san raffaele scientific institute, milan, italy introduction: microglia is considered an eligible target against the progressive multiple sclerosis (ms), but currently available therapies do not allow its efficient targeting. as many cell types, microglia communicate with the neighbouring cells through a complex system of extracellular vesicles (evs) exchange. recently my group described that microglia derived-evs, engineered to encapsulate il4, are taken up by microglia itself, mediating a phenotype switch to a protective phenotype. in vivo studies suggest that these evs can ameliorate established neuroinflammation, thus making them a promising drug-delivery tool to target cns in ms. my project focuses on understanding the mechanism of action and the signalling pathway of evs delivery and to exploit this knowledge to specifically deliver different potential therapeutic molecules. for this purpose, we decided to characterize the evs through trps technology. methods: a murine microglia cell line (bv2) was engineered to stably overproduce endogenous il4. this cell line was cultured in exosome-depleted rpmi and stimulated with pma(20 mg/ml) for 30 min. evs isolation was carried out by collecting supernatant and subjecting it to consequential centrifugation of 300 g,10 min, rt and 2000 g,20 min, 4°c. the resulting supernatant was filtered (5 µm) and ultracentrifuged at 100,000 g for 2h at 4°c. the evs pellet was re-suspended in ice-cold pbs. the evs analysis with trps shows two populations of evs, one with a mean diameter of 60-80 nm and a broad zeta potential ranging from −10 mv to −60 mv, while the second population has a mean diameter of 120-140 nm and a zeta potential of −10/-20 mv. this difference can be consistent with the different pathway formation of exosomes and microvesicles. we demonstrated in vivo the strong phenotypic change induced by our evs to resting microglia in a dose-and time-dependent effect. then, impairing the physiological procedure of the endosome acidification, the effect of our evs on recipient cells is higher. thus, suggesting an endocytic pathway for the internalization of the vesicles. we further demonstrate with gradient ultracentrifugation the capability of our formulation to vehicle endogenous il4 inside the vesicles. even if some protein is co-purified in the procedure, we know that the half-life of this cytokine is too short to elicit a strong in vivo response. consequently, we assume that the anti-inflammatory effect of our evs in vivo is a result of the il4 internalized in our formulation. summary/conclusion: these data help us understand more in detail the process of internalization and phenotype change mediated by these evs. our next goals are to discriminate between different internalization pathways and further validate the efficacy of our therapy on the eae mouse model. targeting il-3rα on tumour-derived endothelial cells blunts metastatic spread of triple negative breast cancer via extracellular vesicle reprogramming introduction: the lack of an approved targeted therapy and the early onset of metastasis highlight the need for new treatments for triple-negative breast cancer (tnbc) patients. interleukin-3 acts as an autocrine factor for tumour-endothelial-cells (tec), and exerts pro-angiogenic paracrine action via extracellular vesicles (nevs). il-3rα blockade on tec changes tec-ev (anti-il-3r-evs) microrna cargo and promotes the regression of established tumour vessels. as tec are the doorway for "drug" entry into tumours, we have aimed to assess whether il-3r blockade on tec impacts tumour progression via their unique ev cargo. methods: 27 human tnbc samples, mda-mb-231, mda-mb-453 and mcf10 cell lines were evaluated for the expression of il-3rα. nevs and anti-il-3r-evs were characterized by electron-microscopy, macsplex-exosome-kit and western blot. proliferation, migration, apoptosis and sphere formation were evaluated. scid mice were used for in vivo experiments. results: we noticed that, besides tec and inflammatory cells, tumour cells from 55.5% of the human tnbc samples expressed il-3rα. mda-mb-231 and mda-mb-453, but not mda10 cells, expressed il-3rα. in vitro, nevs provide survival and migratory signals, while anti-il-3 r-evs promoted apoptosis as well as reduced cell viability and migration of human tnbc cell lines. in vivo anti-il-3 r-ev treatment induced vessel regression in established tumours formed of mda-mb-231 cells and almost abolished the spread of liver and lung metastasis. moreover, decreased β-catenin and twist1 were found in tumours from animals treated with anti-il-3 r-evs. in addition, anti-il-3 r-evs reduced lung metastasis that was generated via the intravenous injection of mda-mb-231 cells. nevs that were depleted of mir-24-3p (antago-mir-24-3p-evs) were effective as anti-il-3 r-evs in down-regulating twist1 and reducing lung-vessel density and metastatic lesions in vivo. summary/conclusion: overall, these data provide the first evidence that il-3 rα is highly expressed in tnbc cells, tec and inflammatory cells, and that il-3 rα blockade on tec impacts tumour progression. introduction: high-grade serous ovarian cancer (hgsoc) is the deadliest gynaecologic cancer. its lethality is explained for late diagnosis at advanced stages and frequent recurrences despite achieving complete response with standard therapy. most of recurrences occurs at abdominal cavity with multiple metastasis. therefore, identifying key determinants of metastatic process remains as priority to find better therapies. current evidence assigns a central role of the exosomes in conditioning the metastatic niche in epithelial cancers. recently, we demonstrated that statins reduce metastasis in hgsoc in preclinical models. here, we decided to study the effects of statins on hgsoc-derived exosomes and its capability to condition the metastatic niche. methods: exosomes were isolated from heya8 ovarian cancer cell line and primary tissue cultures established from advanced-stage hgsocs (with signed informed consent and irb approval) by differential ultracentrifugation and quantified by nanoparticle tracking analysis (nta). enriched-cancer initiating cells (cic) spheroids were established from heya8 cells by using stem-selecting conditions. the paracrine effect of exosomes on cic migration/invasion was studied using either 3d migration or boyden chamber invasion assays. previous to exosome isolation, heya8 cells were treated with simvastatin (5um, 24 h) or solvent and proteins involved in exosome biogenesis/uptake (alix, tsg101), its trafficking (rab7a, rab27a) and in conditioning the metastatic niche (emmprin) were measured by immunoblotting. results: exosomes isolated from heya8 cells or hgsocs enhance the metastatic potential of heya8established spheroids in 3d migration or boyden chamber invasion assays. upon simvastatin treatment, we observed a significant reduction in migration/invasion induced by equivalent number of exosomes in heya8-derived cics. under same treatment, we observed a significant decrease in protein levels of alix and tsg101 and an increase in the inactive forms of rab7a and rab27a in heya8 cells. we also observed a decrease in emmprin levels in heya8-derived exosomes. summary/conclusion: here, we demonstrated a paracrine effect of hgsoc-derived exosomes that favour the metastasis process. in addition, we demonstrated that simvastatin reduces metastasis induced by cancerderived exosomes. such an effect is partially explained by changes in the expression of proteins involved in exosome biogenesis/uptake, its endocytic trafficking and in the content of proteins conditioning the metastatic niche. thus, simvastatin arises as potential therapeutic target to improve outcomes in this disease. funding: this research was supported by fondecyt granted to mauricio a. cuello label-free optical imaging and characterization of cancer-associated extracellular vesicles in tissues introduction: cancer-associated extracellular vesicles (evs) visualized in the tumour microenvironment have been identified as a potential biomarker for cancer-related tissue changes. analyses of evs have traditionally been performed in cells or isolated evs, with no temporal or spatial information that could be critically important for elucidating their roles in carcinogenesis. since the unperturbed distribution and organization of evs in the tumour microenvironment is associated with their cellular function and can potentially serve as a diagnostic and prognostic biomarker, there is a strong need for visualizing evs in freshly isolated tissue specimens. currently, only fluorescent labelling methods enable visualization and tracking of evs. we used a custom label-free multimodal multiphoton optical imaging system to detect and characterize evs and classify them using their optical signatures both in isolated tissues and in situ tumours. methods: label-free multimodal multiphoton imaging was used to provide simultaneous, co-registered structural and functional images of evs in untreated samples. heterogeneous populations of evs could be identified from their unique optical signatures. results: the intrinsic metabolic and structural properties of evs enabled reliable visualization and optical characterization of evs from cell cultures and in situ imaging of tumour-bearing rats. unique optical signatures were then used for identification of cancer-related evs in tissues from human breast cancer patients, and their density was found to be highly correlated with clinical diagnosis. in the current study, evs were isolated from urine of tumour-bearing dogs, and urine and serum from breast cancer patients. analysis of ev content showed higher concentration of nad(p)h in evs isolated from cancer subjects, than from healthy subjects, which reflects the reprogramming of cellular metabolism in carcinogenesis. summary/conclusion: these results suggest a potential label-free optical methodology to detect and characterize evs by their optical signatures, which can be utilized as possible diagnostic and prognostic biomarkers for cancer. funding: this research was conducted under protocols approved by the iacuc and irb at the university of illinois and carle foundation hospital, and supported by funding from nih. novel potential anticancer therapies based on interference with nuclear entry of cancer cell-derived extracellular vesicle components in recipient cells introduction: the intercellular communication mediated by extracellular vesicles (evs) in the tumour microenvironment plays an important role in tumour progression. experimental evidence indicates that evs derived from highly metastatic cells influence the behaviour of less aggressive cancer cells. we have previously described a novel intracellular pathway where a fraction of endocytosed ev-associated proteins and nucleic acids is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into nucleoplasmic reticulum (nr). here, we better characterize this pathway and report that it is required for the induction of an aggressive behaviour induced by evs released from highly metastatic sw620 colon cancer cells in isogenic primary cancer cells. methods: super resolution-structured illumination microscopy and magnetic-based co-immunoisolation studies were employed to identify the protein components of the nuclear pathway and to monitor the entry of ev-containing late endosomes into the nucleoplasmic reticulum. human sw480 carcinoma cells expressing er-gfp and rab7-rfp were exposed to evs from sw620 cells and then live imaged. results: we have previously reported that the tripartite protein complex, containing vap-a, orp3 and rab7 orchestrates the localization of ev-carrying late endosomes into nr. we now report that silencing of orp3 or vap-a, but not its homologue vap-b, reverses the pro-metastatic changes induced by evs isolated from metastatic cells on their non-metastatic counterpart, including transition to an ameboid phenotype, cell rounding and blebbing. moreover, we found that certain nuclear pore complex proteins and importin-beta1 are co-immunoisolated with orp3, vap-a and rab7 suggesting the formation of a large protein complex at the entry of nuclear pores. summary/conclusion: interfering with the mechanisms regulating this novel intracellular pathway may find therapeutic applications particularly in ev field and oncology. educated osteoblasts regulate breast cancer proliferation via small extracellular vesicles thomas jefferson university, philadelphia, usa introduction: breast cancer commonly traffics to bone, where breast cancer cells (bccs) can survive undetected for years before metastatic outgrowth. in bone, bccs interact with surrounding stromal cells, including osteoblasts (obs), to shape the metastatic niche. our lab discovered there are at least two subpopulations of obs in the bone-tumour niche, based on protein marker expression. one group, "educated osteoblasts" (eos) have engaged in crosstalk with bccs whereas another group, naïve obs, have not. we have novel evidence that eos regulate bcc proliferation. the purpose of this study was to determine if extracellular vesicles (evs) produced by eos play a role in regulating bcc proliferation. we hypothesized evs produced by eos would decrease bcc proliferation. methods: eo-derived small evs from culture media were isolated via ultracentrifugation and characterized evs for size, protein marker expression, and density floatation to validate the purity of ev samples. the functionality of eo-derived evs on bcc proliferation was examined using edu and checkpoint proteins p21 and p27. bcc protection from chemotherapy induced cell death was also examined. results: we found that evs produced by eos, but not naïve obs, decreased both triple negative and erpositive bcc proliferation in a concentration dependent manner. furthermore, using an edu assay, we found that exposure to eo-derived evs induces bccs to undergo cell cycle arrest. interestingly, the cell cycle arrest was reversible and bcc proliferation was restored upon removal of eo-derived evs. in addition, exposure to eo-derived evs leads to increases in bcc expression of the g1 checkpoint proteins, p21 and p27. we next wanted to investigate proliferative signalling pathways that may be deregulated in bccs following exposure to eo-derived evs. we found that eoderived evs reduce bcc levels of erk1/2. because our data indicate eo-derived evs induce sustained cell cycle arrest in bccs, we desired to know if eo-derived evs protected bccs from chemotherapy-induced cell death. we found that bccs exposed to eo-derived evs and the chemotherapy drug, doxorubicin, have decreased cell death compared to bccs exposed only to doxorubicin. summary/conclusion: altogether, our data suggest eos play a crucial role in bone-tumour microenvironment by regulating bcc proliferation. funding: supported by nih r00 ca178177 and commonwealth of pennsylvania -department of health sap 4100072566 for kmb. phosphorylation of tyrosine 23 in annexin a2 is essential for its association with exosomes and for imparting invasive and proliferative capacity to other cells priyanka prakash desai a , pankaj chaudhary b , xiangle sun b and jamboor vishwanatha a a unt health science center at fort worth, fort worth, usa; b unt health science center, fort worth, usa introduction: triple negative breast cancer (tnbc) accounts for 15%-20% of all breast cancer cases. the lack of targeted-based therapies highlights the importance of studying tnbc. elevated levels of annexin a2 (anxa2), a ca+2-dependent phospholipid binding protein, has been correlated with worse overall survival in tnbc patients. our previous data implicate that exosomal anxa2 is involved in creating a pre-metastatic niche and facilitating metastasis in tnbc. moreover, n-terminal phosphorylation of tyrosine (tyr) 23 in anxa2 has been implicated in regulating several anxa2 activities in cancer progression. here, we demonstrated that n-terminal phosphorylation of anxa2 at tyr23 is important for its association with exosomes which imparts invasive and proliferative phenotype to other cells. hence, dissecting the regulatory pathway will be critical for verifying the value of anxa2 as a therapeutic, diagnostic or prognostic marker in tnbc. methods: pn1-egfp plasmids expressing the constitutive phosphomimetic (anxa2-y23e) and non-phosphomimetic mutant (anxa2-y23 f) gene expressing mutation at tyr23 site were overexpressed in mda-mb-231 tnbc cells. mutant cells were experimentally validated for anxa2 specific functions like migration, invasion and proliferation. exosomes were isolated from the mutant phosphomimetic (exo-anxa2-y23e-gfp) and non-phosphomimetic (exo-anxa2-y23 f-gfp) cells and were analysed for exosomal surface expression of anxa2 by immunoprecipitation and flowcytometry. cal-148 breast adenocarcinoma epithelial cells were treated with exo-anxa2-y23e-gfp and exo-anxa2-y23 f-gfp to analyse the rate of invasion and proliferation by transwell invasion and proliferation assay, respectively. transfer of exosomal anxa2 in cal-148 was studied using immunofluorescence and its implications on signalling pathways were studied by western blot. results: mda-mb-231 phosphomimetic tnbc mutant cells showed increased migratory, invasive and proliferative capacity compared to non-phosphomimetic tnbc mutant cells. exo-anxa2-y23e-gfp had elevated surface anxa2 expression compared to exo-anxa2-y23 f-gfp. cal-148 cells treated with exo-anxa2-y23e-gfp showed high migratory, invasive and proliferative characteristics, with a higher expression of p-anxa2(tyr23), p-src(tyr416) and p-paxillin(tyr31) compared to exo-anxa2-y23 f-gfp treated cells. summary/conclusion: n-terminal phosphorylation of tyr23 in anxa2 in mda-mb-231 tnbc cells (phosphomimetic mutant cells) is essential for its association with exosomes and for conferring increased invasive and proliferative capacity to other breast cancer cells. funding: the above study is funded by national institute of health ro1ca220273 and nimhd's u54md006882 to dr.j.k.vishwanatha. a novel method for epithelial-derived extracellular vesicle isolation and enrichment in patients with advanced prostate cancer arpit rao, helene barcelo and bharat thyagarajan university of minnesota, minneapolis, usa introduction: evaluation of changes in prostate cancer biology is difficult due to presence of lymph nodal or bony metastatic disease in a majority of patients. a number of liquid biopsy assays have shown clinical utility in prostate cancer, but are limited by low sensitivity (e.g. circulating tumour cells-based assays) or inability to perform transcriptome sequencing (cellfree dna-based assays). epithelial-derived extracellular vesicles (epi-ev)-based assays are uniquely positioned overcome both these limitations as evs are abundantly secreted into the blood and have rnacargo that mirrors the cell of origin. however, a reliable method to enrich for epi-evs is currently lacking. methods: plasma was isolated from the peripheral blood collected from 15 patients with metastatic prostate cancer enrolled in an institutional biobanking study before initiation of systemic antineoplastic therapy. evs were isolated from 500 µl of plasma using a commercially available qev 35 size exclusion column (izon inc.). without subjecting the evs to any physical stressors such as centrifugation, cd61 magnetic beads were used to fractionate the evs into cd61+ (platelet derived) and cd61-(non-platelet derived) fractions. multiparameter flow cytometry was used to evaluate evs that expressed cd9 and epcam and were negative for calnexin. nanotracking analysis (nta) was used to quantify both total ev and cd61+ and cd61fractions in all patient samples. the average ± standard deviation of total evs obtained from the 15 patients was 6.39x10^11 ± 4.60x10^11 evs/ml of plasma (coefficient of variation [cv] : 72%) while the average and standard deviation of cd61-evs was 1.24x10^10 ± 3.37x10^10 (cv: 271%). the cd61-ev fraction represented a variable amount of the total evs in prostate cancer patients ranging from 0.0001% to 32.21%. multiparameter flow cytometry showed that over 80% of total evs were cd9+ and calnexin-, suggesting an endosomal origin for a vast majority of the evs in these plasma samples. however, the proportion of evs expressing epcam (marker of epi-evs) was higher among the cd61-fraction (5% -30%) as compared to the cd61+ fraction (0.04% -1%). summary/conclusion: our novel method was able to isolate and enrich the epi-ev from the plasma of advanced prostate cancer patients. correlation between clinical characteristics and ev quantity is being evaluated to identify the reason(s) for large variations in cd61-ev fraction. future studies are planned to use our method in improving the sensitivity of ev-based assays and increase the rna yield to facilitate transcriptome sequencing. funding: this work was funded by grants from randy shaver community and research fund, minnetonka, mn. exosomes drive medulloblastoma metastasis in a mmp2 and emmprin dependent manner introduction: recurrent/metastatic medulloblastoma (mb) is a devastating disease with an abysmal prognosis of less than 10% 5-year survival. the secretion of extracellular vesicles (evs) has emerged as a pivotal mediator for communication in the tumour microenvironment during metastasis. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the blood-brain-barrier. matrix metalloproteinases (mmps) are enzymes secreted by tumour cells that can potentiate their dissemination by modification of the extracellular matrix. we hypothesise that exosomal mmp2 and its inducer emmprin could enhance metastasis of mb. methods: proliferation, invasion and migration assays were used to evaluate the phenotypic behaviour of primary cell lines pre-treated with metastatic tumour cell-derived exosomes. gelatin zymography and western blotting were performed to confirm mmp2 functional activity in cell lines and exosomes. nanoscale flow cytometry was used to measure surface exosomal emmprin levels. exosomal mmp2 and emmprin were modulated at the rna level. results: number of exosomes is directly related to the migratory behaviour of parental mb cell lines (p < 0.01). notably, functional exosomal mmp2 and emmprin levels also correlate with this. furthermore, exosomes from metastatic cell lines conferred enhanced migration and invasion on their matched isogenic primary (non-metastatic) cell line pair bỹ 3.8-fold (p < 0.01). exosomes from metastatic cell lines also conferred increased migration on poorly migratory foetal neuronal stem cells. summary/conclusion: together this data suggests that exosomal mmp2 and emmprin may promote medulloblastoma metastasis and supports analysis of exosomal mmp2 and emmprin levels in patient cerebral spinal fluid samples. introduction: exosomes secreted from cancer cells harbour the potential to regulate intracellular signalling and promote metastasis. wherein, metastasis suppressor genes (msgs) play a pivotal role in regulating such signalling cascades. however, the regulation gets hampered due to low expression of msgs under metastatic conditions. nm23-h1, product of first identified metastasis suppressor gene nme1, is significantly downregulated under metastatic conditions. nm23-h1 serves as a regulator of small gtpases. several evidences have highlighted an involvement of small gtpases (such as rab5, rab7 and rab27) in the biogenesis of exosomes. in addition, bacterial homolog of nm23 has been shown to interact with rab5 and rab7. however, experimental evidence supporting a relationship between exosomes and nm23-h1 is lacking. our current focus is to deduce the relationship between exosomes and msgs. methods: breast cancer cell lines were used to assess the effect of exosomes isolated from highly metastatic cells (mda-mb-231 cells) on lower/non metastatic cells (mcf-7 cells). nme1 was overexpressed in mda-mb-231 cells and subsequently used to isolate exosome fractions. equivalent amount of isolated exosome fractions from mda-mb-231 cells and mda-mb-231/nme1 cells were utilized to access their effect on migration and difference in exosome markers. results: we observed an enrichment of nm23-h1 in the exosomes isolated from mda-mb-231 cells upon overexpression of nme1. proteinase k protection assay confirmed the packaging of nm23-h1 inside the exosomes isolated from mda-mb-231/nme1 cells and excluded the possibility of membrane association of nm23-h1. additionally, overexpression of nm23-h1 led to a significant reduction in the ability of mda-mb-231 exosomes to stimulate movement of mcf-7 cells as confirmed by wound healing assays. our data also highlights a clear reduction in the protein levels of exosome markers such as cd63, cd9 and alix in the exosome fraction isolated from mda-mb-231/nme1 cells as compared to mda-mb-231 cells. interestingly, rab7a, a protein involved in the endosome-lysosome fusion was also present in lower amount in the exosomes isolated from nm23-h1 overexpressing cells. summary/conclusion: our data highlights an antimigratory effect of nm23-h1 via exosomes. these findings support a regulatory role of nm23-h1 in the packaging or release of exosomes in highly metastatic breast cancer cells, and further suggest that metastasis suppressor proteins may be involved in the regulation or packaging of exosomes. additional studies will be required to decipher the downstream signalling of nm23-h1 which affects the biogenesis of exosomes as well as to assess the effect of nm23-h1 overexpression on the content of exosomes. these insights could help us delineate the complex exosome biogenesis pathway and provide new potential drug targets for exosome regulation. introduction: exosomes (exs) are emerging as novel players in the beneficial effects induced by exercise on vascular diseases. our recent study has revealed that moderate exercise enhances the function of circulating endothelial progenitor cell-derived exosomes (cepc-exs) on protecting endothelial cells against hypoxia injury. in this study, we aimed to investigate whether exercise-regulated cepc-exs contribute to the beneficial effects of exercise on ischaemic stroke (is). methods: c57bl/6 mice performed moderate treadmill exercise (10 m/min, 4-wks) before is induced by middle cerebral artery occlusion surgery. acute injury was evaluated at day 2 by determining neurologic deficit, infarct volume, cell apoptosis in the penumbra and neurologic recovery was assessed by determining angiogenesis/neurogenesis, sensorimotor functions at day 28. the correlations of cepc-exs and their carried mir-126 with neurological parameters were analysed. the underlying mechanism of the effects of cepc-exs isolated from exercised mice was explored in a hypoxia neuron model. cellular mir-126 level, apoptosis, axon growth ability and gene expressions (cas-3, bdnf and akt) were measured. results: 1) exercised mice had a smaller infarct volume on day 2, which was associated with decreased cell apoptosis and cleaved cas-3 level, and a higher microvessel density than those in control; 2) the elevated cepc-ex level positively correlated with tepc-exs in ischaemic brain of exercised mice on day 2. the upregulated mir-126 level positively correlated with the numbers of tepc-exs in ischaemic brain; 3) the numbers of cepc-exs and their carried mir-126 level negatively correlated with the infarct volume, cell apoptosis and positively correlated with the microvessel density in the peri-infarct area on day 2; 4) exercised mice had decreased infarct volume, increased microvessel density, promoted angiogenesis/neurogenesis and improved sensorimotor functions on day 28, accompanying with upregulated levels of bdnf, p-trkb/trkb and p-akt/akt; 5) cepc-exs of exercised mice protected neurons against hypoxia-induced apoptosis and compromised axon growth ability which were blocked by mir-126 and pi3 k inhibitors. summary/conclusion: our data suggest that the protective effects of moderate exercise intervention on the brain against mcao-induced ischaemic injury are ascribed to cepc-exs and their carried mir-126. funding: this work was supported by american heart association (18post33990433) and nih (1r01ns 102720). syndecan-1 regulates alveolar type 2 epithelial cell senescence mediating through extracellular vesicles during lung fibroproliferation tanyalak parimon a , changfu yao a , adam aziz a , stephanie bora a , marilia zuttion1 zuttion a , dianhu jiang a , melanie koenigshoff b , cory hogaboam a , paul nobel a , barry stripp a and peter chen a a cedars-sinai medical center, los angeles, usa; b university of colorado, denver, usa introduction: alveolar type 2 epithelial cell (at2) senescence is implicated in the pathogenesis of lung fibrosis, a progressive fatal condition. syndecan-1, a heparan sulphate proteoglycan, is overexpressed by at2 cells of human idiopathic pulmonary fibrosis (ipf) and bleomycin-injured wt mice and the overexpression of syndecan-1 is profibrotic. moreover, syndecan-1 deficient (sdc1-/-) mice had less lung fibrosis after bleomycin injury. we reported that extracellular vesicles (evs) in bronchoalveolar lavage (bal) of bleomycin-injured wt mice augmented lung fibrosis whereas the sdc1-/--bal-evs attenuated the process. moreover, wt-bal-evs expressed lower level of anti-fibrotic mirnas (mir-34b-5p, −142-3p, −144-3p, and −503-5p) compared to the sdc1-/-bal-evs. these mirnas targeted genes in the cellular senescence pathway indicating that syndecan-1 altered microrna profiles in the bal-evs to promote cellular senescence during lung fibrogenesis. we investigate how syndecan-1 regulates at2 senescence through evs. methods: bleomycin was intratracheally given into wt and sdc1-/-mice. at day 21, lungs were processed for single-cell rna sequencing (scrnaseq) and western blot (wb). evs were isolated using ultrafiltration centrifugation method. human (a549) and mouse (mle-15) lung epithelial cell lines were used for in vitro experiments. results: scrnaseq analysis indicated while bleomycin stimulated an overexpression of cellular senescencespecific genes on at2 cells of wt mice, these genes were significantly downregulated on sdc1-/-at2 cells. senescence proteins, p16 and p21, were also less expressed in the lungs of sdc1-/-than of the wt mice by wb. to determine the functional effects of evs in bal, a549 cells were treated with human ipf or control lung wash-evs and evaluated for beta-galactosidase activity. we found that ipf-evs markedly increased beta-galactosidase enzymatic activity. corroborating with these data, bleomycin-injured bal-wt-evs also significantly upregulated senescence marker, p21, by wb on mle15 cells whereas sdc1-/--bal-evs inhibited p21 expression. summary/conclusion: our data indicate that syndecan-1 regulates lung fibrosis through the senescence signalling pathway on at2 cells. furthermore, syndecan-1 controls at2 senescence mediating through extracellular vesicles in the bal. lastly, the most likely cargo molecules mediating this process are micrornas. immortalized cardiosphere-derived cell ev-associated pirna, imev-pi, protects against ischaemic injury in the heart alessandra ciullo, ahmed ibrahim, liang li, chang li, weixin liu and eduardo marbán smidt heart institute, cedars sinai medical center, los angeles, usa introduction: cardiosphere-derived cells (cdcs) are a population of heart-derived progenitors with demonstrated therapeutic efficacy in preclinical and clinical settings. cdcs function by secreting extracellular vesicles (evs), lipid-bilayer nanoparticles laden with bioactive molecules. recently our group developed a strategy for immortalizing cdcs (imcdc) that retains their therapeutic potential and enhances cdc function indirectly through their secreted evs. imcdc show a different rna content(mirna, mrna, rrna, trna and pirna) compared to primary cdc. in particular, we focus on piwi rnas (pirnas), small rnas bound by piwi proteins, important regulators of both the epigenome and transcriptome. we seek to explore the role of a pirna highly enriched in imcdc-evs (imev-pi). methods: evs are prepared by conditioning cells for 24 hrs in serum-free basal media, in hypoxic culture. after 24 hrs conditioned medium is cleared of cellular debris and evs isolated using ultrafiltration by centrifugation (ufc). fractions were analysed in terms of particle size, number, and concentration and pirna content. in vitro, bone marrow derived-macrophages (bmdm) were exposed to imcdc-ev, imev-pi and control and transcriptomic profile and potentially activated pathways were assessed. in vivo, 8-10 week-old wistar-kyoto female rats received 10^10 imcdc-ev, imev-pi, scramble or vehicle intracoronary 20 minutes after ischaemiareperfusion(i/r). cardiac troponin i levels, scar size and monocytes were assessed at 24 and 48 hrs. results: by small-rna sequencing analysis we found that pirnas are enriched in both cdc-ev and imcdc-ev. imcdc show a different pirna composition compared to primary cdc. imev-pi was identified as one of the most highly-expressed non-coding rnas (the number of reads were 35x higher in imcdc-ev compared to cdc-ev). in vitro, imexo-pi-conditioned bmdm exhibit a different transcriptomic profile compared with control, with upregulation of pathways involved in the inflammatory response, cell death, and cell-to cell signalling. in vivo, imev-pi is cardioprotective, as shown by reduced scar size and lower cardiac troponin levels compared to vehicleand scramble-injected animals at 48 hrs post i/r. imev-pi only minimally alters neutrophil counts profile in blood but it alters monocytes profile with a decreased number at 24 hrs and an increase at 48 hrs. summary/conclusion: we posit that imev-pi is a key determinant of imcdc-ev therapeutic efficacy. our results indicate that target cells may be macrophages/ monocytes, given that imev-pi exposure modifies their composition and mrna profile both in vitro and in vivo. introduction: extracellular vesicles (exosomes, evs) are cell membrane particles (30-200 nm) secreted by virtually cells. during intercellular communication in the body, secreted evs play crucial roles by carrying functional biomolecules (e.g., micrornas and enzymes) into other cells to affect cellular function, including disease progression and tissue regenerations. literature previously reported that the macropinocytosis pathway contributes greatly to the efficient cellular uptake of evs. the activation of growth factor receptors, such as epidermal growth factor receptor (egfr), induces macropinocytosis. in this study, we demonstrated the effects of evs on demal papilla and hair follicle regeneration. methods: identification of distinct nanoparticles and subsets of extracellular vesicles from umbilical cord blood stem cell by asymmetric flow field-flow fractionation. results: the effects of evs from umbilical cord blood stem cell on the propagation of demal papilla and hair follicle regeneration were observed. summary/conclusion: the enhancement of extracellular vesicles from umbilicalcord blood stem cell the propagation of demal papilla and hair follicle regeneration were observed and confirmed. mechanisms of host resistance to plasma membrane damage induced by pneumolysin attack introduction: bacterial pore-forming toxins (pfts) are major virulence factors produced by pathogens. pfts target host plasma membrane (pm) and create transmembrane pores, which allow uncontrolled flux of ions and small molecules across the pm disrupting cellular homoeostasis. to survive, cells display poorly understood repair mechanisms to recover the cell homoeostasis. several mechanisms were proposed to participate in cell recovery: exocytosis of cortical lysosomes; endocytosis of pfts pores; pm blebbing and shedding. methods: we used increasing concentrations of purified ply to intoxicate cells. pm permeability was assessed by flow cytometry using propidium iodide dye. cytoskeleton rearrangements were investigated by confocal immunofluorescence microscopy. extracellular vesicles released during pm repair were isolated by high-speed centrifugation and characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and mass spectrometry/ liquid chromatography analysis. results: ply triggers a complete reorganization of the actomyosin cytoskeleton inducing the formation of cortical actomyosin bundles at sites of pm remodelling. these structures assemble upon loss of pm integrity and disassemble as pm recovers. we detected the release of microvesicles during the recovery of pm integrity. vesicle population is heterogeneous with sizes ranging from 100 to 500 nm, with the majority of them measuring 100-200 nm. vesicle proteomic analysis revealed that they contain ply, suggesting they participate in pore removal, proteins involved in vesicle trafficking, pm repair and exosome biogenesis. summary/conclusion: our data demonstrate that cells are able to recover from the damage induced by sublytic concentrations of ply. actomyosin cytoskeleton undergo massive changes with the assembly of cortical bundles possibly at sites of pm damage. we showed that cells produced extracellular vesicles during the process of repair. we are now focusing on understanding the biogenesis of those vesicles and its importance during the process of repair. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms (2d static tissue culture flask vs 3d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in 2d t-flasks and 3d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both 2d and 3d culture systems. msc-ev were characterized according to misev 2018 guidelines, using techniques as nta, protein and lipid quantification, western blot, imaging and fourier-transform infrared spectroscopy (ftir). the results indicate that msc-ev derived from different sources/donors have similar size distribution, however, ev yields tend to be higher for the 3d culture system. of notice, several spectral regions were identified by ftir, enabling the detection of differences in the biomolecules present in msc-ev, msc-conditioned media and cells produced under different conditions. summary/conclusion: in summary, this study contributes to the establishment of a scalable process for msc-ev production. evaluation of three different isolation methods for small extracellular vesicles from human plasma in prostate cancer diagnosis introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies. methods: pca patients and age-matched healthy controls (hc) plasma (n = 6 in each group) were used to isolate sevs with 3 different isolation methods including commercial exoquick ultra kit, qev 35 and qev 70 size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd9 and cd81 commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination. results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev35 demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev70. furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods. summary/conclusion: qev 70 method demonstrated better performance in isolating relatively pure sevs from human plasma; qev35 has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev35 but with the highest non-sev protein contaminations. people can choose higher sev content or higher sev purity according to the downstream analysis. moreover, sevs may also be used for treatment monitoring, as recent studies suggested that the expression levels of certain markers may change during therapy, reflecting tumour response. for cancer diagnostics and therapeutic purposes in clinical settings, it is important to have a device which allows multiplexed measurements, in order to scan a large number of markers simultaneously and compare the expression levels of different patients, or same patients at different treatment stages, in a time efficient manner. methods: herein, we propose a multiplexed platform for label-free detection and surface protein profiling of sevs. the technique is based on the electrokinetic phenomena of streaming current and zeta potential (\zeta*) and measures the\zeta* change upon sev binding on functionalized microcapillary surfaces. for the purpose, we used sevs derived from lung cancer cells. in its current form, the platform can measure up to 5 channels simultaneously, however, it can be further expanded. results: having demonstrated that our electrokinetic sensor successfully detects sevs in a specific way, we tested its ability to measure the expression level of membrane proteins. the analysis showed that it could detect differences in the expressions of egfr on sevs, with a sensitivity of 10%. we then extended the platform for multiplexed analysis, by connecting and measuring four capillaries, functionalized with different capture probes, simultaneously. for the purpose, we targeted specific tumour markers, i.e. egfr, and exosomal tetraspanin family proteins, such as cd9 and cd63. the results showed successful multiplexed ev detection. summary/conclusion: being the sensor suitable for multiplexed sev detection, we shall present our investigation on a set of pleural effusion samples collected from a cohort of lung-cancer patients with different genetic makeup. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd9, cd63 and cd81, it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: 1) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and 2) the assessment of subpopulation target enrichment relative to the population average by leveraging tim4 as an unbiased, lipid-based ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasis-associated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr3 (her2+), t47d and mcf-7 (er+pr+), bt549 and mda-mb-231 (triple negative) breast cancer cell lines, as well as five mda-mb-231-derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her2 was broadly detected in her2+ skbr3 evs. interestingly, her2-t47d and mcf-7 evs also expressed her2 where it was highly enriched in its epcam+ subpopulations. itg α6, β3 and β4 were only found in triple negative and organotropic evs with itg β3 and β4 differentially enriched based on the organotropism. the population average of mda-mb-231 and lung-tropic evs had high expression of itg β4, where subpopulations of cd44+ evs showed positive enrichment while cd9+ and cd63 + evs showed negative enrichment. itg α5, β3 and β4 were absent in the bone-tropic cd81+ ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb-231 evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du145 prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified 49 targets that bind to hs-gag chains, and also 108 different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of ev-associated hspgs will result in attenuation of ev induction of a tumour-supporting fibroblast phenotype. introduction: ovarian cancer (oc) is the fifth leading cause of cancer-related death in women, partly due to difficulty in early diagnosis. extracellular vesicles (evs) show promise for use in early diagnostics of oc. here, evs from cervical mucus (cm) of ovarian cancer patients were used for discovery of oc biomarkers for diagnostics. machine learning was used to mine ev mirna data to develop an oc biomarker panel (validation via the cancer genome atlas). examination of the mirna targets reveal that the panel is a sufficiently accurate predictor of oc. methods: evs from the cm of 48 patients (15 highgrade serous, 24 low-grade, 7 benign) were isolated for small rna-sequencing. the top differentially expressed mirnas were used in a random forest and "voom" (variance modelling at the observational level) model. unsupervised approaches were used and then vetted against patient symptomology data. a tcga ovarian cancer dataset (n = 100) was used for validation. results: an oc biomarker panel of 10 micrornas (voom: 96.55% accuracy; random forest: 88% accuracy) was generated. the panel consists of members from the mir-200 family and the mir-16 family, among others. the mirna targets are associated with molecular functions and pathways specific in oc progression. summary/conclusion: our method has identified ev mirna biomarkers that may be crucial for early, noninvasive detection of oc. data science has been used to develop a feedback system integrating biochemical experiments, smaller datasets, and previously available data to identify and verify a biomarker panel for oc diagnostics. introduction: liver disease has become a significant cause of morbidity and mortality among hiv patients. alcohol exposure can further exacerbate liver damage by activating hepatic stellate cells (hscs), leading to hepatic fibrosis or cirrhosis, often seen at all levels of alcohol exposure among people with hiv. due to the potentiating effects of alcohol on hiv-induced hepatocytes (hep) damage, as well as the effect of ethanol in hsc-mediated extracellular remodelling, it is imperative to understand the interplay of hep and hscs. here, we focus on the exosomes released by hiv-and ethanol exposed hep and how these exosomes modulate the functional behaviour of hscs. methods: human hepatocyte huh7.5cyp2e1 [hepatoma cells stably transfected with cyp2e1 designated as rlw cells] were infected with hiv in the presence or absence of alcohol metabolite, acetaldehyde using the acetaldehyde-generating system (ags). the conditioned medium was collected from 4 groups of cells: untreated, hiv-, ags-and hiv+ags. quantification of exosomes number and size were evaluated with zetaview or nanosight and further characterized for exosome markers following the guideline from minimal information for studies of evs 2018 (misev2018). the human hepatic stellate lx-2 cell line was exposed to hepatocyte-derived exosomes and assessed for the activation using pro-inflammatory markers il-1β, il-6, tnfα, and fibrotic markers acta2, and timp1 using quantitative pcr. we also analysed exosome mirna content in primary human hepatocytes (phh), which potentially regulates the function of recipient cells by "programming" their inflammation/fibrosis status. the network analysis for mrna and mirna were carried out using gene ontology consortium, and mirror 2.0 and david bioinformatics resources 6.8. results: ags treatment further enhanced the release of hiv-induced exosome from hepatocytes. size distribution assessed by zeta view or nanosight revealed that approximately 85-90% of particles distributed in the range of 50 to 200 nm, with a peak at~90 nm. enriched expression of hiv protein p24 was observed in fractions f9-f12. western blotting of hepatocytederived exosome demonstrated positivity for exosome-enriched proteins alix, tsg 101 and cd9 specifically in f2-f8 fractions and negative for endoplasmic reticulum protein calnexin. the uptake of hepatocytederived exosomes by hscs was apparent as demonstrated by immunofluorescence. the internalization of hepatocyte-exosome induced activation of hscs as evidenced by increased expression of pro-inflammatory il-1β, il-6, tnfα markers in the latter cells. summary/conclusion: we conclude that ags treatment in hiv-infected hepatocytes potentiates the release of exosomes, which, following uptake by the hscs, leads to their activation. funding: this work is supported by nih-1 r01 aa027189-01a1. antimicrobial peptide ll-37 induces neutrophil-derived extracellular vesicles with antibacterial potential and protects murine sepsis yumi kumagai, taisuke murakami, kyoko kuwahara and isao nagaoka juntendo university, bunkyo-ku, japan introduction: extracellular vesicles (evs) released from immune cells or other host cells upon microbial infection modulate the immune response and thereby regulate the infection. sepsis is a life-threatening multiple organ dysfunction caused by systemic dysregulated inflammatory response to infection. nevertheless, numerous therapeutic trails concerning immune dysfunction have still been disappointing outcomes. we have previously shown that ll-37, a human cathelicidin antimicrobial peptide, improves the survival of caecal ligation and puncture (clp) septic mice. here, we investigated the induction of ev release by ll-37 and functions of ll-37-induced evs in murine sepsis. methods: evs were isolated from peritoneal exudates of clp mice and the supernatant of ll-37-stimulated mouse bone marrow neutrophils by differential centrifugation or size exclusion chromatography. isolated evs were analysed by flow cytometry, western blotting, and nano particle analysis. neutrophil-derived evs were injected into clp mice to assess the protective function of evs against septic mice. the antibacterial activity of evs was evaluated by incubating with escherichia coli. results: in clp mice, ll-37 augmented the level of evs. evs from ll-37-injected clp mice contained higher amounts of neutrophil-derived antibacterial proteins (lactoferrin and cramp, cathelicidin-related antimicrobial peptide) and exhibited higher antibacterial activity compared to evs from pbs-injected clp mice. furthermore, ll-37 stimulated mouse bone marrow neutrophils to release evs with antibacterial potential, and administration of the ll-37-induced evs reduced the bacterial load and improved the survival of clp mice. summary/conclusion: ll-37 induces the release of antimicrobial evs from neutrophils in clp mice, thereby reducing the bacterial load and protecting mice from lethal septic condition. identification of mirna profiles of serum exosomes in active tuberculosis introduction: tuberculosis (tb) has exceeded hiv as the most lethal infectious disease globally for two consecutive years, mainly due to difficulties in achieving early and definitive diagnosis, and timely treatment. exosomes carrying rna, particularly mirna, have demonstrated their functional and diagnostic potential in diseases including tb. however, few published studies have explored whether exosomal mirnas could be used for diagnosis of tb. thus, more systematic and comprehensive study of exosomal mirnas with regard to their potential as non-invasive tb biomarkers is still urgently needed. methods: we searched the gene expression omnibus database for datasets published before december 2019, and performed meta-analysis on available exosomal mirna profile data for healthy control (hc) and active tb clinical specimens . reprocessing next generation sequencing data under uniform parameters and utilizing state-of-the-art bioinformatics analysis. results: we identified many distinct up-regulated and down-regulated differentially expressed exosomal mirna across multiple studies, and further screened the top 10, which might provide a potential panel for differentiation of hc and tb. we classified all differentially expressed mirnas into six expression patterns and identified two persistently up-regulated mirna (hsa-mir-140-3p, and hsa-mir-423-3p) as potential markers during tb progression. moreover, the differential expressed exosomal genes that we screened from the datasets were consistent with the genes overlapped with predicted mrna targets of differentially expressed mirna. pathway and function analysis further demonstrated down-regulated signalling pathways/immune response and up-regulated metabolism and apoptosis/necrosis. introduction: trypanosoma cruzi is a protozoan parasite that causes chagas disease, a relevant source of morbidity in latin america, which has spread to many countries as result of immigration of the people from endemic areas. many studies have been showed that trypomastigote forms of t. cruzi release extracellular vesicles (ev) that increase parasite infection. objectives. here, we aim to test if previous immunization with evs in adjuvant can generate a protective immune response by decreasing the effects of evs in experimental chagas disease. methods: female balb/c mice were immunized by intra peritoneal (ip) administration with 5 × 105 or 107 evs isolated from trypomastigotes forms, with aluminium hydroxide adjuvant (aloh). injections were administered intravenous in 3 doses during 45 days (15 days interval). after immunization, mice were infected intra-peritoneally with 500 trypomastigotes forms. parasitaemia was quantified by counting motile parasites in fresh blood sample drawn from lateral tail veins. mortality and weight were analysed during the infection. in control group, the mice were immunized with aioh. results: the immunization with evs with aloh decreased the blood parasitaemia and the animals survived, while all animals died in the group aloh alone. the animals immunized with evs had an increase of f4/80+ cd11b+ and cd80/cd86 expression in cells isolated from the peritoneum. summary/conclusion: these results indicate that t. cruzi ev antigens can induce an immune response that controls the development and establishment of the experimental chagas disease. introduction: acinetobacter baumannii (ab) is a nosocomial pathogen, of major concern due to its multidrug resistance (mdr) and the recent appearance of hyper-virulent strains in the clinical setting. the world health organization included ab as a critical priority pathogen for the development of novel antibiotics. ab pathogenesis is associated with a multitude of potential virulence factors (vf) that remain poorly characterized. there is growing evidence that outer membrane vesicles (omv) are used as vehicles to transport bacterial proteins that contribute to set up the conditions for the infections. in the present work we studied the physiopathology of mdr ab. we focused on the contribution of non-characterized outer membrane proteins (omps) associated to omvs, with special focus on lipoproteins (lp). methods: we conducted a bioinformatic prediction using available datasets to construct a list of omv-associated omps putatively acting as vf in ab5075. seven genes were selected and the corresponding mutants were obtained from manoil lab collection. physiological analyses of the mutants were performed, and the involvement of the selected proteins in ab pathogenesis was evaluated by adherence, invasion, and cytotoxicity assays on human lung cells a549. results: biochemical analysis indicated similar growth rates in rich media, as well as similar levels of omv production for all the mutants as compared to wt. also, no differences in susceptibility to chaotropic agents were observed, indicating no alteration of the om function as a general permeability barrier. all mutants similarly reduced a549 cell viability, but to a lesser extent than the wt. moreover, three of them exhibited less adhesion and invasion compared to the wt, and omv isolated from these mutants displayed variable levels of cytotoxicity. summary/conclusion: these results suggest roles for the mutant gene products in ab pathogenesis and contribute to the better understanding of ab virulence mechanisms, revealing novel possible targets for therapeutic development. funding: agencia nacional de promoción científica y tecnológica (anpcyt, pict 2017-3536) medicine, nanfang hospital, southern medical university, guangzhou, 510515, china, guangzhou, china (people's republic); d zhujiang hospital, southern medical university, guangzhou, china, guangzhou, china (people's republic) introduction: talaromyces marneffei (t. marneffei) grows as a mycelial form in the environment but multiplies rapidly as a yeast form in the host and within macrophages. the yeast can cause disseminated and progressive infections or lethal talaromycosis. but the mechanisms of pathogenicity of t. marneffei are poorly understood. fungal extracellular vesicles (evs) have previously been shown to transmit a proinflammatory message to macrophages. however, the characteristics and effects of t. marneffei evs on the progress of infection have not yet been investigated. methods: in this study, evs of t. marneffei yeasts were isolated by ultracentrifugation method. evs were detected and confirmed by electron microscopy and nanoparticle tracking analysis (nta). the raw 264.7 murine macrophages were incubated with the t. marneffei vesicles to observe the changes of macrophage morphology and function, especially in inflammatory response. the proteins, dnas, rnas of t. marneffei vesicles were respectively removed with protease, dnase and rnase. all treated evs were used to incubate with murine macrophages observe the effect on macrophages in inflammatory response. results: we observed that evs secreted by t. marneffei have a typical spherical shape with a diameter of 30 to 200 nm. t. marneffei evs were internalized by raw 264.7 murine macrophages and promoted the production of no and proinflammatory cytokine by macrophages in a dose-dependent manner. t. marneffei evs stimulate macrophages to generate reactive oxygen species (ros). addition of t. marneffei evs to macrophages also promoted transcription of the m1-polarization marker cd86 and diminish that of the m2 markers cd206. incubation of t. marneffei vesicles with murine macrophages resulted in increased levels of extracellular interleukin-1β(il-1β), interleukin-6 (il-6) and interleukin-12 (il-12). the proinflammatory effect of vesicles was weakened when the proteins of the vesicles were destroyed. in contrast, no similar changes were observed in degraded dna and rna. summary/conclusion: our results indicate that the extracellular vesicles of t. marneffei can stimulate macrophage towards to m1 polarization phenotype and promote proinflammatory function. plasma-derived extracellular vesicles as potential biomarkers in chronic chagas disease patients introduction: chagas disease (cd), caused by the parasite trypanosoma cruzi (t. cruzi), is a neglected tropical disease affecting about 8 million people worldwide. currently, one of the main clinical problems is the lack of effective biomarkers for therapeutic response and disease prognosis during chronic infections. in that context, extracellular vesicles (evs) are raising attention as novel, minimally invasive, and inexpensive method for diagnostic and screening of diseases, as well as a new source to identify new biomarkers. the main objective of this study is to use evs derived from biological fluids of chronic cd patients for identifying novel biomarkers, specifically in the context of therapeutic response and disease prognosis. methods: plasma, saliva and urine from a cohort of chronic cd patients are being collected before and at the end of benznidazole treatment. as negative controls, healthy donors have been also included. the purification and characterization of the evs was performed by size exclusion chromatography, followed by nanoparticle tracking analysis, bead-based flow cytometry assay and transmission electron microscopy. a proteomic analysis of the evs was also performed. results: proteins associated with evs secreted by infective t. cruzi have been previously identified in cell culture, but never in human samples. our results, based on the analysis of a single heart-transplanted patient with chronic cd, showed the presence of t. cruzi and human proteins specifically associated with plasma-derived evs. noticeably, several human and parasite proteins identified in evs obtained from plasma samples, were present or upregulated before chemotherapy and were absent or downregulated following treatment. currently, proteomics analyses are being performed with higher numbers of cd plasma samples. summary/conclusion: to the best of our knowledge, this is the first proteomic profiling of plasma-derived evs from a heart-transplanted patient with chronic cd. these results thus open the possibility of using evs from biological fluids as a tool for the identification of new biomarker candidates in chronic cd. these biomarkers are essential for assessing disease introduction: eukaryotic cells communicate with one another through multiple pathways. an established route of communication between eukaryotic cells is via the production of a range of different membrane bound signalling "packages", called extracellular vesicles (evs). evs are produced by all domains of life and carry proteins, nucleic acid (rna and dna), and other biological material, travelling between cells and around the body to deliver a range of chemical messages. bacteria can also produce evs that communicate with each other to coordinate population behaviour, as well as with eukaryotic cells to stimulate host defence or induce tolerance. here i investigate the poorly explored axis where evs are the vehicle for communication between eukaryotic cells and bacteria. methods: as a first step, i have isolated evs from tissue cultured eukaryotic cells grown in advanced rpmi media with minimal ev-depleted fbs. nanoevs were isolated from spent culture media using sequential centrifugation (2,000 × g, 10,000 × g) and concentration (100 kda filter) before purifying using size exclusion chromatography columns. nanoev-rich fractions were pooled based on particle (nanoparticle tracking analysis) and protein quantity data. nanoevs were characterised by electron microscopy and expression of exosomal markers. eukaryotic nanoevs were then characterised in their effect upon the growth of escherichia coli as a model bacterium, also grown in tissue culture media to mimic relevant in vivo conditions. results: further experiments with increased dosages are required to determine the effect of human evs on bacteria. summary/conclusion: our work will investigate whether human evs communicate with the resident and pathogenic microbiota, while examining the mechanisms behind this communication. escherichia coli pathogenic bacteria commensal bacteria hydrogen sulphide (h2 s) derived extracellular vesicles: a potential protective role in response to respiratory syncytial virus (rsv) infection methods: evs were isolated from untreated (control evs) and gyy4137 treated (gyy-evs) a549 cells, a human alveolar type ii-like epithelial cell line. evs were purified using a two-step enrichment procedure. evs were characterized using particle sizing (size and concentration) and western blot for the ev markers. electron microscopy and immunofluorescence staining were used to investigate presence of multivesicular bodies (mvbs), evs precursors, in both groups. recipient a549 cells were cultured for 24 hours in the presence or absence of control-or gyy-evs, then infected with rsv for 24 hours. viral titres by plaque assay were measured in recipient infected a549 cells. results: we confirmed the presence and purity of our evs. we found that gyy4137 reduced the particles number of evs, but did not change ev size. a549 cells treated with gyy4137 showed an accumulation of mvbs/lysosomes-like structures, as well as an increase in cd63 expression, a mvbs marker, compared to untreated cells. recipient a549 cells treated with gyy-evs showed lower viral replication than control ev-treated cells in response to rsv infection. we are currently investigating the potential mechanism for this observation and characterizing the rna cargo composition of gyy-evs. summary/conclusion: no vaccine or effective treatment is currently available for rsv. cellular pretreatment with gyy-evs reduced the rsv replication in airway epithelial recipient cells, suggesting that h2 s could exert its antiviral activity in the context of rsv infection potentially through modulation of ev composition. therefore, gyy-evs could represent a future novel pharmacological approach for ameliorating virus-induced lung disease. effects of extracellular vesicle-mediated transmission on reoviridae infection results: taken together, these data suggest that multiple particles of reovirus and rotavirus egress in large, virus-modulated evs, and that transmission in evs increases segment complementation compared to transmission as free particles. summary/conclusion: these discoveries may be broadly applicable to viruses that travel in evs and will contribute to general principles of virus transmission and diversification. continued studies will illuminate the specific cellular pathways reovirus and rotavirus utilize for successful egress. these pathways may prove to be critical targets for the improvement of vaccines and oncolytic therapy. multiparameter flow cytometry analysis of the human spleen and its interaction with plasma-derived evs from plasmodium vivax patients introduction: the spleen is a secondary lymph organ that filters blood and elicits immune responses against blood-borne pathogens, such as malaria parasites. extracellular vesicles (evs) are membrane-bound particles involved in intercellular communication. evs play several roles in malaria ranging from modulation of immune responses to induction of vascular alterations. here, we report the first integrated characterization of human spleen cells using multiparameter flow cytometry (mfc) describing subpopulations of splenic leukocytes and red blood cells (rbcs), and studied their interaction with plasma-derived evs from p. vivax patients (pvevs). methods: human spleens were obtained from organ transplantation donors. myeloid, lymphoid, erythroid and haematopoietic stem cells (hscs) were immunophenotyped by mfc. t cells, dendritic cells (dcs) and rbcs were enriched by density centrifugation and immunomagnetic isolation. pvevs and healthy donors evs (hevs) were purified by size-exclusion chromatography (sec) and characterized by bead-based flow cytometry. enriched evs were labelled with fluorescent lipophilic dyes and incubated with total splenocytes or enriched populations. evs-cells interaction was assessed by flow cytometry. results: human spleen immunophenotyping showed that cd45+ cells included b (30%), cd4 + t (16%), cd8 + t (10%), nk (6%) and nkt (2%) lymphocytes. myeloid cells comprised neutrophils (16%), monocytes (2%) and dcs (0.3%). erythrocytes represented 70% whereas, unexpectedly, reticulocytes were 0.93% of total cells. in addition, we also detected hscs, which accounted for 0.02%. sec separated evs from the bulk of soluble plasma proteins as shown by the enrichment of cd63, cd5 l and cd71 markers. interaction studies showed an increased proportion of t cells (cd4 + 3-fold and cd8 + 4-fold), monocytes (1.5-fold) , b cells (2.3-fold) and erythrocytes (threefold) interacting with pvevs as compared to hevs. summary/conclusion: the integrated cellular analysis of the human spleen and the methodology employed here allowed in vitro interaction studies of human spleen cells and evs. a larger proportion of monocytes, t and b lymphocytes as well as erythrocytes was found to interact with pvevs compared to hevs. future functional studies of these interactions can unveil pathophysiological processes involving the spleen in vivax malaria. neuroblastoma-secreted exosomes carrying mir-375 promote osteogenic differentiation of bone marrow mesenchymal stromal cells introduction: bone marrow (bm) is the major target organ for neuroblastoma (nb) metastasis and its involvement is associated with poor outcome. yet, the mechanism by which nb cells invade bm is largely unknown. tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (mscs) have been recognized as a fundamental part of the associated tumour stroma. here, we explore the potential role of nb-derived exosomes in induction of a pro-osteogenic phenotype on bm-mscs. introduction: extracellular vesicles (evs) are nanosized particles delimited by a lipid bilayer which transfer functional molecular cargos from the cells of origin to target cells. this intercellular crosstalk controls both physiological and pathological conditions. given their presence in body fluids and their characteristics, these nanocarriers might be potentially used in diagnostics and/or therapy. breast cancer is the most frequently diagnosed malignancy and ranks as the leading cause of cancer mortality in women worldwide; the triple negative breast cancer, in particular, is the most aggressive subtype with a poor prognosis. since it is recognized that cell stiffness of cancer cells play a crucial role during the metastatic spreading, we set ourselves the goal of clarify the effects and the activity of small-evs (i.e. with a diameter below 200 nm) in metastatic breast cancer, with a special attention on their correlation with the biomechanical properties of cells. methods: functional assays were performed on the non-invasive mcf7 breast cancer cell line, before and after the cellular uptake of small-evs originating from the invasive mda-mb-231 triple negative breast cancer cell line. the mechanical properties (cell stiffness, cytoskeleton organization and focal adhesions) of mcf7 cells were investigated before and after the vesicle uptake. results: the uptake of small-evs derived from mda-mb-231 significantly reduces the young's modulus values of mcf7 cell line making them more invasive. moreover actin and focal adhesion variations were observed in mcf7 cells before and after small-ev's uptake, suggesting a molecular rearrangement inside mcf7 cells upon uptake. summary/conclusion: our results evidence that small-evs play a key role in altering biomechanical properties of target cells and underline their relevance in cell-cell crosstalk. our approach is very promising to identify new molecular mechanisms through which evs perform their oncogenic function. stratification of angiogenic or non-angiogenic lesions in colorectal cancer liver metastases patients using extracellular vesicle mirna introduction: colorectal carcinoma (crc) is the second leading cause of cancer death in the western world. over 50% of the crc patients develop liver metastasis (lm) and 90% will die from metastatic disease. in the current clinical setting, liver resection provides the only possible cure, but only 20% of crclm patients are resectable. the combination of angiogenic inhibitors with chemotherapy is used to downsize crclm with the goal of converting unresectable patients to resectable ones. however, only 10-15% of these patients can be successfully converted to a resectable state. we have no way of identifying those crclm patients that would respond/benefit to the addition of anti-angiogenic therapies (e.g. bevacizumab: bev)). proper stratification of patients into angiogenic inhibitor responders and non-responders will permit a proper assessment of the efficacy of angiogenic inhibitors. crclm forms 2 distinct histopathological growth patterns (hgp): angiogenic (desmoplastic) and nonangiogenic (replacement) hgp. we demonstrated that crclm patients with predominant angiogenic lesions receiving bev plus chemotherapy have a more than double 5-year overall survival compared to patients with non-angiogenic lesions. therefore, nonangiogenic lesions do not respond to angiogenic inhibitors. our study focuses on stratifying angiogenic vs non-angiogenic lesions of crclm through extracellular vesicle mirnas. we are using two approaches in the selection of mirnas to target: 1. text mining of published ev mirna from crclm patients; and 2. differentially expressed mirnas present in tumour tissue from both lesion types, we have obtained by sequencing 50-60 patients. these two strategies will generate a list of mirnas that we will target using qpcr on plasma-derived ev mirna from the patients used in approach 2, where we have classified the lesions in the patients. preliminary data on 10 patients will be presented. methods: ev isolation was performed using the gold standard centrifugation method. rnaseq and qpcr are used to generate the expression profile for angiogenic vs non-angiogenic type of crclm. results: the research is under progress. summary/conclusion: the research is under progress. the introduction: it is known that bone metastasis causes a reduction in the quality of life of cancer patients due to fractures and nerve compression. therefore, it is important to elucidate the mechanism of bone metastasis and develop new treatments. metastatic bone tumours occur at particularly high rates in cancers of the prostate, breast, and lung. in this study, we focused on extracellular vesicles (evs) in bone metastasis, and investigated that the role of evs derived from cancer cells in osteolysis. methods: the prostate, breast, and lung cancer cellderived evs were added to osteoclast precursors with rankls. the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase (trap) stain and by measuring the expression level of osteoclast markers using by qrt-pcr. a proteome analysis (lc-ms/ms) and sirna approaches were used to identify molecules which are responsible for promotion of osteoclast differentiation in the prostate cancer cellderived evs. to investigate whether the molecules are suitable for the detection of bone metastasis in serum evs, we isolated evs from serum of prostate cancer patients, and analysed the protein level of the molecules by western blot analysis. results: we found that the prostate cancer and lung cancer-derived evs significantly promoted the rankl-stimulated osteoclast differentiation. our analysis revealed that cub domain-containing protein 1 (cdcp1), which is a membrane protein on the prostate cancer cell-derived evs, was responsible for promotion of osteoclast differentiation. moreover, cdcp1 was markedly detected in the evs-derived from serum of prostate cancer patients who had bone metastasis than that of normal subjects. we also found that cdcp1 exits on the breast and lung cancer cell-derived evs. summary/conclusion: we showed that the evsderived from bone metastatic tumours have a role in activation of osteoclastogenesis. moreover, we revealed that cdcp1 in the evs is responsible for promoting of osteoclast differentiation. these evs could be the novel diagnostic and therapeutic target for bone metastasis. increased expression of chemokine receptor cxcr4 in non-invasive colorectal cancer cells after incorporation of platelet-derived extracellular vesicles. introduction: blood platelets and platelet-derived extracellular vesicles (p-evs) play a crucial role in tumour growth and metastasis. p-evs, also referred to as platelet microparticles, are recognized as a carrier for proteins and nucleic acids that control cell-to-cell communication, mediate the formation of metastatic niches and affect tumour invasion and metastasis. among the other factors, p-evs contain the chemokine receptor cxcr4, known as a co-receptor for hiv entry but also regarded as important in cancer development due to the importance of cxcr4/cxcl12 signalling. overexpression of cxcr4 was reported in various, especially in invasive cancers, including colorectal cancer (crc). crc, the third most commonly diagnosed cancer, is usually diagnosed at the late stage and patient's death is mainly related to metastasis. increased levels of cxcr4 has been reported as a poor prognostic factor for survival of crc patients and its blocking has been suggested as therapeutic approach. the aim of this study was to analyse the effect of p-evs on the levels of cxcr4 in crc cells on various epithelial-to-mesenchymal transition stage. methods: we used crc cell lines ht29 and sw620, which represent distant invasive potential and different phenotypes, epithelial and strongly mesenchymal, respectively. p-evs were isolated from outdated concentrates of human blood platelets after activation by thrombin in the presence of calcium ions, by subsequent centrifugation and ultracentrifugation. the p-evs were labelled using pkh67 fluorescent dye to visualize their uptake into cell lines by confocal microscopy. we also quantified the levels of cxcr4 in ht29 and sw620 by western blot analysis. the effect of p-evs uptake on the migration of crc cells was studied by "wound healing" method. results: we found that the levels of cxcr4 in crc lines used in the study were correlated with their emt stage. we show here that p-evs released by activated platelets were incorporated into both ht29 and sw620 cell lines. the expression of cxcr4 in ht29 was increased after the uptake of p-evs. additionally we observed that migration rate of ht29 cells with incorporated p-evs was elevated as compared to control cells. summary/conclusion: we posit that circulating p-evs can be incorporated into yet not invasive crc cells to significantly increase the level of cxcr4 receptors and that may lead to the their more invasive characteristics. introduction: for cancer therapy it is important to identify markers and key processes induced during cancer progression. one of them is epithelialmesenchymal transition (emt) which is associated with cell acquisition of invasiveness, stem cell characteristics and resistance to apoptosis and therapy. also the extracellular vesicles (evs) released from tumour cells, which can be taken up by cells constituting pre-metastatic niches, can alter cancer progression by promoting cells' reprogramming. our group has recently reported that snail transcription factor, a key factor of emt, when overexpressed in crc ht29 cells, drives their early emt and alters the expression of microrna (mirs). in the present study we analysed the mirs profile of evs released from those cells. methods: evs from three ht29 clones stably overexpressing snail and from control ht29-pcdna were isolated by differential centrifugation and ultracentrifugation of conditioned media after 24 h of culturing in serum-free medium. total rna was isolated and nextgeneration sequencing (ngs) analysis of the mirnas was performed followed by gene ontology ( introduction: prostate cancer (pca) is the most common malignant tumour in male urinary system and osteoblastic bone metastasis is the most observed metastasis in prostate cancer patients. it has been demonstrated that circulating micrornas contained in extracellular vesicles are potential early biomarkers and therapy targets for many diseases. however, the potential role of micrornas in prostate cancer bone metastasis, is not yet to be fully explored. methods: after isolation and purification evs using ultracentrifugation from conditioned media of bone metastatic co-opting prostate cancer cells and normal cells, total rna was extracted. subsequent to library preparation and small rna-seq, differential gene expression analysis was performed. data were filtered by mean mirna expression of ≥ 50 reads, two fold up or down regulation between 2.5 − 7.5 and adjusted pvalue ≤ 0.05. the uptake of pca-sevs was performed. three candidate mirnas (has-mir-200 c-3p; has-mir-1275; has-mir-383-5p) were internalized and osteoblast differentiation were detected by qpcr, histochemical staining and protein activity detection. results: total reads of mirnas in bone metastatic co-opting pca-evs exceeded significantly than that in normal evs (p < 0.001), indicating that mirnas delivered by pca cells play critical role in pca bone metastasis. pca-cm enhanced osteoblast differentiation and can be reversed by gw4869. the uptake of pca-evs by mc3t3-e1 was efficient. the high expression of the three candidate mirnas in pca-evs was verified by qpcr. all the three candidate mirnas promoted osteogenesis, verified by mrna expression of osteoblastic markers (alp, ocn, runx2, osx), alp activity, alp staining and aliza red s staining. summary/conclusion: these findings suggest that mirna cargos in pca-evs play a pivotal role in the development of osteoblastic bone metastasis of pca, which can be potential early biomarkers and therapy targets for prostate cancer bone metastasis. funding: this work was supported by grants from the national natural science foundation of china (81872347); xijing hospital science and technology foundation project (xjzt19ptk14). introduction: retinoblastoma (rb) is the most common intraocular cancer of childhood. despite recent advances in conservative treatment have greatly improved the visual outcome, local tumour control remain difficult in presence of massive vitreous seeding. thus, the identification of new biomarkers is crucial to design more effective therapeutic approaches. traditional biopsy has long been considered unsafe in rb, due to the risk of extraocular spread. exosomes, nano-sized vesicles containing nucleic acids and proteins, represent an interesting alternative to detect tumour-associated biomarkers. the aim of this study was to determine the protein signature of exosomes derived from rb tumours (rbt) and vitreous seeding (rbvs) primary cell lines. methods: exosomes from 4 rbt (hsjd-rbt1, hsjd-rbt2, hsjd-rbt5, hsjd-rbt14) and 3 rbvs (hsjd-rbvs1, hsjd-rbvs3, hsjd-rbvs10) cell lines were isolated by high speed ultracentrifugation. vesicles number and size were confirmed by nanosight and scanning electron microscopy. protein content was analysed by bicinchonic-acid assay and high resolution mass spectrometry. results: a total of 5666 proteins were identified. among these, 5223 and 3637 were expressed in exosomes rbt and one rbvs group respectively. gene enrichment analysis of exclusively and differentially expressed proteins and network analysis identified identified in rbvs exosomes upregulated proteins specifically related to invasion and metastasis such as proteins involved in extracellular matrix (ecm) remodelling and interaction, resistance to anoikis and metabolism/catabolism of glucose and aminoacids. summary/conclusion: in conclusion, in this study, we isolated exosomes from rb primary tumour and vitreous seeding cell lines and characterized their content with a proteomic approach. this is the first evidence describing a proteomic exosome signature specifically associated with vitreous seeding in rb. this characterization may represent a starting point for future analyses that allow defining exosomal markers as promising diagnostic and potential prognostic markers in rb as well as therapeutic targets. activation of hepatic stellate cells by extracellular vesicles released by uveal melanoma cells introduction: uveal melanoma (um) is the main intraocular tumour in adults, and is particularly resistant to treatments when disseminated to the liver. our hypothesis is that extracellular vesicles (evs) released by the primary tumour are priming the liver stroma for metastatic cell colonization by activating hepatic stellate cells (hstecs). this study aimed to characterize evs from um cells, and to determine their interactions with liver cells. methods: evs were isolated from cell lines derived from ocular tumours and liver metastases by differential centrifugation. their concentration/diameter range were determined by high-sensitivity flow cytometry. cryo-tem combined with receptor-specific gold labelling was used to reveal the morphology/size of melanomic evs. the presence of melanoma and ev markers was assessed by western blotting. the internalization of fluorescent melanomic evs in hstecs and their subsequent activation were assessed by confocal imaging using alpha-smooth muscle actin (alpha-sma) and phalloidin stainings. ev impact on invasion was measured with a tumour spheroid model embedded in extracellular matrix. melanomic evs were inoculated into the retro-orbital sinus of immunodeficient mice to study their selective organ distribution. results: melanomic evs were positive for annexin-5, tetraspanins, as well as some melanoma markers. stellate cells with internalized melanomic evs expressed more alpha-sma, reflecting their activation. adding evs on tumour spheroids increased the invasion process. melanomic evs were localized into different murine organs, but mainly into the liver, as observed by in vivo fluorescent imaging. introduction: exosomes are being tested for their use as therapeutic agents in degenerative and chronic diseases. however, the optimal source of exosomes is currently under investigation. amniotic fluid (af) is a naturally-rich source of exosomes that is easily obtained for use in regenerative medicine. organicell flow™ is a minimally-manipulated, acellular product derived from human af and consist of over 300 cytokines/chemokines as well as exosomes derived from the amniotic membrane and surrounding tissues. we characterized the exosome fraction of our product to elucidate the protein cargo of af exosomes and demonstrate the therapeutic potential as a novel regenerative therapy. methods: the exosome fraction of our product was analysed using nanosight nanoparticle imaging and macsplex exosome surface marker array analysis. exosomes were precipitated using size-exclusion filtration followed by ultracentrifugation from 3 independent products (in triplicate) and subjected to protein lysis and preparation for mass spectrometry analysis using the easy nlc 1000 and q exactive instruments. tune (version 2.9) and xcalibur (version 4.1) was used to collect data while proteome discoverer (version 2.2) was used to analyse data. protein expression lists were created by merging the 3 sample replicates together and commonly expressed proteins were determined using vinny 2.0 vin diagram analysis. webgestalt tool kit classification system was used to identify top protein function and pathway hits. results: organicell flow™ contain a mean concentration of 5.24x10^11 particles/ml (n = 12) with a mean mode size of 125.2nm (n = 12). surface marker analysis confirms the presence of exosome associated proteins cd63, cd81, and cd9 in addition to a high expression of cd133 (n = 3). the completed analysis revealed 1225 commonly detected proteins across 3 products. the top molecular functions of identified proteins included protein-binding, ion-binding, and nucleic acid-binding with enzymes, transcription regulators, and transporter proteins representing the most abundant protein groups. pathway enrichment analysis revealed top hits for integrin, pdgf, and p53 pathways. a deeper dive into the enzyme category of the protein cargo further demonstrates the presence of proteins that promote dna repair such as dna polymerase (beta and lambda), telomerase reverse transcriptase, and brca1. summary/conclusion: organicell flow™ characterization demonstrates the therapeutic potential of afderived exosomes. proteomic analysis revealed protein cargo that may regulate various growth factor and cellcycle associated pathways. furthermore, the presence of dna damage response proteins suggests a possible mechanism for induction of cellular repair. generation of car-t and γδt cell-derived exosomes for future cell free immunotherapies γδt cells are a subset of t cells with dual innate and adaptive qualities. this duality provides various advantages over their more studied and used counterpart, αβt cells. in the present study, we sought to compare the immunotherapeutic potential of car-t cell and γδt cell-derived exosomes as novel cell-free based alternatives. methods: cd19-targeting car-t cells were obtained following the isolation, expansion and transduction of αβt cells using a lentiviral vector bearing the car construct. γδt cells were isolated and expanded from peripheral blood mononuclear cells (pbmcs) following innate or adaptive stimulation. exosomes from both cell sources were isolated after a 3-day culture in serum-free media using ultracentrifugation-based methods. exosomes were characterized by nanoparticle tracking analysis (determination of size) and western blot assays (detection of the appropriate surface markers). nalm-6 (b cell precursor leukaemia) cells were used as target cells for assessment of exosome cytotoxic/ killing function. car-t cell and γδt cell-derived exosomes were incubated at 1000 particles/target cell for 24-hours. total viable cell counts were assessed via imaging-based cytometry (nc-3000) utilizing acridine orange and dapi staining. results: exosomes derived from γδt cells activated via innate mechanisms showed significant killing of nalm-6 as compared to exosomes from non-activated or adaptively activated γδt cells. in comparison, car-t cell-derived exosomes showed minor killing capabilities of the target cells. summary/conclusion: here, we report for the first time that exosomes derived from cd19 car-t cells and innately activated-γδt cells show/exert inhibitory action on nalm-6 cells. further studies are currently underway to identify the underlying mechanism(s) responsible. introduction: age-related cognitive dysfunction is associated with increased oxidative stress, low-level chronic neuroinflammation, and waned hippocampal neurogenesis in the brain. from this perspective, biologics capable of modulating oxidative stress and neuroinflammation, and stimulating neural stem cell activity in the brain might be useful as anti-ageing interventions. methods: we investigated the efficacy of intranasal administration of extracellular vesicles (evs) generated from cultures of rat subventricular zone neural stem cells (svz-nscs) in the middle-aged mice to alleviate cognitive and mood dysfunction, increased oxidative stress, neuroinflammation, and neurogenesis decline in old age. mice were treated intranasally with nsc-evs once weekly for three weeks (50 billion per administration) starting from 18.5 months of age. a month later, the animals were examined for cognitive, memory, and mood function using multiple behavioural tests, and brain tissues were examined for oxidative stress, neuroinflammation, and neurogenesis. results: object-based tests revealed that aged animals receiving vehicle displayed cognitive impairments for discerning minor changes in the environment as well as for distinguishing similar but not identical experiences. these animals also exhibited spatial memory dysfunction and anhedonia. in contrast, aged animals receiving nsc-evs showed improved cognitive and mood function. biochemical analyses of brain tissues revealed that nsc-ev treatment normalized elevated concentrations of oxidative stress markers malondialdehyde and protein carbonyls and the proinflammatory cytokine interleukin-1 beta. moreover, nsc-ev treatment stimulated increased production of antiinflammatory protein interleukin-10 and the antioxidant superoxide dismutase. immunohistochemical analysis revealed modulation of neuroinflammation typified by reduced activity of reactive astrocytes and activated microglia and improved hippocampal neurogenesis. summary/conclusion: the results suggest that the intranasal administration of nsc-evs is a promising approach for maintaining better cognitive and mood function in ageing through modulation of oxidative stress, neuroinflammation, and neurogenesis. funding: supported by a grant from the national institute of neurological disorders and stroke (1r01ns106907-01 to a.k.s.) chemically modified myocytes-derived evs for the treatment of cardiac fibrosis. marta prieto-vila a , asao muranaka a and takahiro ochiya b a tokyo medical university, tokyo, japan; b tokyo medical university, shinjuku-ku, japan introduction: myocardial fibrosis is a disorder that may occur after cardiac injure due to a malfunction of the cardiac remodelling. fibroblasts resident in myocardium are erroneously activated causing an excessive accumulation of extracellular matrix, which decreases cardiac function and eventually, leads to death. it is known that cardiomyocytes communicate with the surrounding cells such as fibroblast and endothelial cells by extracellular vesicles (evs). the loss of this communication is thought to play a central role in cardiac fibrosis. therefore, cardiomyocytes-derived evs may be a promising a cell-free system for the treatment of fibrosis inhibition. methods: a novel culture medium was stablished to improve the expansion of primary cardiac myocytes. this was tested using two commercially available primary myocytes cell lines. evs were collected by serial ultracentrifuges, and their effect on fibrosis was tested. for that, prior to any treatment, and to mimic fibrosis, primary cardiac fibroblast were activated overnight with tgfβ. results: by the use of a defined conjunct of chemicals, mature cardiomyocytes culture was highly improved to ensure a high collection of evs. terminal differentiation markers, as well as senesce apparition was delayed in comparison to predetermined culture medium. interestingly, those primary cells secreted a rather large amount of evs, which expressed common evs membrane marker. tgfβ-treated cardiac fibroblasts were co-cultured with myocytes showing a decrease of fibroblast activation markers both at mrna and protein levels. similar results were found when activated fibroblast were treated with evs. summary/conclusion: our findings indicate that the use of evs derived from chemically modified myocytes is a promising treatment for ischaemic myocardial fibrosis. however, further molecular experiments have to be done to identify the molecules within evs responsible for the inactivation of fibroblast. evaluation of osteoinductive and anti-inflammatory properties of spinederived exosomes renaud sicard a , tania del rivero b , jonathan messer c , shabnam namin c and timothy ganey c a vivex, biologics, inc., miami, usa; b vivex, biologics, inc., miami, usa; c vivex, biologics, inc., miami, usa introduction: over the last 2 decades, mesenchymal stem cell-derived exosomes have been shown to play a crucial role in a myriad of cell function such as extracellular matrix synthesis, proliferation, differentiation or cell migration. biological sources of exosome (heterogeneous or homogeneous cell population, serum, urine etc.) have a direct influence on the content of their cargo and their therapeutic application and potential. in this study, we evaluated exosomes excreted from cadaveric spine-derived cells. we hypothesized that exosomes derived from a bone source such as the spine, will drive the osteogenic differentiation of progenitor cells. we also investigated their effects on inflammation in nucleus pulposus cells using an in-vitro assay. methods: after their isolation and characterization, exosomes derived from cadaveric human spines were assayed for osteoinductive properties. a c2c12 myoblast cell line was treated with different concentrations of exosomes and expression of alkaline phosphatase was measured after 5 days incubation. treatment with bmp-4 was used as positive control. anti-inflammatory properties were assessed by incubating tnf-treated nucleus pulposus cells with exosomes for 3 days. qpcr analysis of mrna expression of inflammatory cytokines (il-6, il1-beta, il-8) metalloproteinases (mmp3 and adamts4), and apoptotic genes (bax, bcl2) was used to determine the effects of exosomes on inflammation. results: spine-derived exosomes positively expressed the exosome flow cytometry markers tested (cd81, cd63 and cd9). the mean number of exosomes per microgram of protein was 3.31 ± 2.33 x 108 indicating a relatively high purity. osteoinductive (oi) testing was performed using different concentrations of exosomes. the oi index of treatment of c2c12 cells with bmp-4, 2 x 108, 1 x 109, 2 x 109, 5 × 109 or 1 × 1010 exosomes alone was 28. 5, 1.0, 3.7, 7.4, 11.8 and 27.6 respectively. anti-inflammatory properties of exosome are currently being assessed and will be presented at the time of the poster presentation. summary/conclusion: administering exosomes alone or in combination with an exogenous scaffold has the potential to repair injured tissue and to restore bone function. the clinical significance of this application is aimed to promote the patients' bone healing process and provide a cell-free therapeutic platform that is safe and effective. administration of human mesenchymal stem cell derived extracellular vesicles modulates the abnormal plasticity of newly born neurons and neuroinflammation in a rat model of status epilepticus maheedhar kodali a , daniel gitai b , dong ki kim a , mariam atobiloye a , bing shuai c , sahithi attaluri c , raghavendra upadhya c , leelavathi n madhu a , olagide w. castro a , darwin j. prockop a and ashok k. shetty c decline in the percentage of newly born neurons displaying basal dendrites. besides, ev treated animals displayed higher percentages of resting microglia (ramified microglia), reduced percentages of activated microglia (microglia expressing iba-1 and cd68), in comparison to animals receiving vehicle after se. interestingly, diminished abnormal plasticity of newly born neurons was accompanied by the preservation of interneurons positive for reelin; a protein believed to guide newly born neurons to their correct locations. summary/conclusion: the results suggest that even a low dose in administration of msc-derived evs after se can limit neurons loss, dampen the abnormal plasticity of newly born neurons, and modulate the activation of microglia. introduction: autism spectrum disorders (asd) are neurodevelopmental disorders characterized by three core symptoms that include social interaction deficits, cognitive inflexibility, and communication disorders. they have been steadily increasing in children over the past several years, with no effective treatment. two percent of all asd patients are suffering from a disorder caused by a mutation in the shank3 gene. shank3 is an important synaptic protein, disruption of this gene directly leads to cognitive and motor impairments. during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. here we test three different autistic mice models. btbr as a multifactorial mice model of autism and two different shank3 mutated mice. the first is a complete deletion of exon 22 (22q13.3) and the second is a specific insertion mutation of guanine to 3680 position in the gene (insg3680) that leads to stop codon. methods: exosomes were isolated using differential centrifugation protocol and characterized using the 2018misev guideline recommendations. each animal received an intranasal administration of 20ul containing 107 exosomes/µl. for intravenous administration, the same number of exosomes, were used, injected in 100µl. results: all three animal models showed significant improvement in their autistic behavioural phenotypes following intranasal administration. the improvement seems to be dose-dependent and was better achieved via intranasal vs intravenous administration. biodistribution of msc-exo showed accumulation in the brain within 72 hours, yet the reduction of the signal was observed in the kidneys, heart and lungs. summary/conclusion: our data suggest that exosomes derived from adipose msc, carry a therapeutic potential in asd, via non-invasive intranasal administration in three different mice models. these data further emphasize our potential therapeutic strategy to reduce symptoms of autism in clinical trials. funding: stem cell medicine ltd. israel. equine tendon injury treatment by evs: an in vitro study introduction: current treatment options for tendinopathies (chronic, painful tendon disorders), are not able to restore the functional properties of native tendons. hence, new treatment options are sought. the efficacy of mesenchymal stem cells (mscs) therapies, which combined with a rehabilitation programme including controlled exercise is the current gold standard in equine tendon treatment, has been shown to be largely due to the cells´paracrine activity. the aim of this study was therefore to evaluate the effect of bone marrow msc derived autologous and allogeneic conditioned medium (cm, full secretome) and their extracellular vesicles (evs) on "tendon healing" in vitro. methods: to compare the "therapeutic" effect of msc derived evs and cm, a standardized scratch assay (wound healing assay) was performed. cm from equine tenocytes, ev depleted medium and medium with or without fcs served as controls. tendons and bone marrow aspirates were obtained from three horses (6, 19 and 23 years) which were euthanized for reasons unrelated to this study. mscs were isolated by ficoll density gradient centrifugation and tenocytes were obtained by migration from tendon explants. for cm and ev production, cells were cultured in ev depleted medium. evs were harvested by a stepwise ultracentrifugation approach and characterized by nanoparticle tracking analysis (nta), western blot (cd9, cd63) and transmission-electron microscopy (tem). results: western blot, nta and tem confirmed successful isolation of evs from equine mscs. the strongest positive effect on wound healing (fastest gap closure) was achieved by msc-cm (p < 0.0071). the gap closure achieved with msc-evs was slower than with msc-cm (p < 0.7985) but faster than with cm of tenocytes (p < 0.8289). donor specific differences in wound healing capability were shown for both autologous and allogeneic application. summary/conclusion: treatment with msc-cm resulted in significantly faster wound healing of adult tenocytes in vitro than msc-evs or tenocyte-cm. mscs donor age shows a significant effect on gap closure following autologous but not allogeneic administration. ev-enriched secretome fraction from gmp-compatible, scalable, human ipsc-derived cardiac progenitors improve heart function in chronic heart failure mice introduction: we have shown that research-use-only grade (res) human ipsc-derived cardiac progenitors (cpcres) can produce a secretome whose small-evenriched fraction (svf) can treat chronic heart failure (chf) in mice. gmp-compatible, scalable processes for a cpc-derived svf suitable for human therapeutic use is needed. methods: ipsc-derived cpc were produced and cultured using gmp-compatible, scalable processes (cpctx). media without cells were "cultured" in parallel for "virgin media" controls (mv). cpcres were cultured as previously described. as a proof of concept, svfs were isolated from conditioned media by ultracentrifugation: cpctx-ev, cpcres-ev and mv. particle size distributions/concentrations (nanoparticle tracking analysis), protein levels (bsa), and the presence of cd-63 (elisa) were determined. in vitro activity was assessed by huvec scratch wound healing assay, and by rat and human cardiomyocyte (cm) survival assays. c57bl/6 mice in chf received echoguided myocardial injection of pbs vehicle control (60ul, n = 11), cpctx-ev (60ul, n = 11), or cpcres-ev (45ul, n = 11). change in cardiac function was assessed by echocardiography. results: cpctx-ev particle sizes were polydisperse (mode~72 nm) at a concentration of~1.6e11 particles/ml (~2,600 particles/cell) and~0.975mu cd63/ug protein. cpctx-ev increased wound healing, human cm survival, and rat cm survival in vitro by 4.75x, 1.4x, and 2x, respectively over mv controls. in chf mice, significantly less cpctx-ev mice, and less cpcres-ev mice had severely progressive heart failure (left ventricular end systolic volume, lvesv, increased >14%) than pbs control mice (pbs vs cpctx-ev, p < 0.05; pbs vs cpcres-ev, p < 0.056), and the average ejection fraction of the pbs group deteriorated 2.5x more than the cpctx-ev group (−4% vs −1.6%, respectively; ns). summary/conclusion: we have a process for cpc differentiation and production of conditioned media suitable for use in human clinical trials from which can be made an svf with the potential to treat chf, possibly through re-vascularization or preservation of cm viability. introduction: exosomes are nanoscale vesicles that mediate cell-to-cell communication via exchanging molecular cargo. mesenchymal stem cell (mscs) modification towards an osteogenic path can occur by uptake of exosomes from other cells. it is less clear whether vesicle placement in the absence of cells will facilitate site-specific delivery through acellular transfer of osteogenic activity. an electrospun fleece was combined with bone marrow-derived exosomes in the absence of cells to evaluate osteoinductive potential that might be thermo-stable and be used in a biologically neutral collagen carrier. comparisons were made of standard laboratory assay of osteoinductivity (oi), and in vivo expression in a mouse calvarial defect model. methods: electrospun type-i collagen was prepared with and without hydroxyapatite (ha) (spinplant gmbh, leipzig) as a foundation base for application of the bone marrow-derived exosomes. individual discs of the collagen enhanced scaffolds (3-mm) were prepared and placed in a mouse calvarial skull defect. animals were followed for 4 and 8 weeks. exosomes were isolated from qualified cadaveric human spines by differential ultracentrifugation. microscopic observation, quantitative assessment of oi with an alkaline phosphatase assay, and flow cytometry were used to evaluate the composition, the hybrid nature of the addition to the nano-collagen fibres. a fluorescent protein reporter transgenic mouse model expressing osteocalcin, type-i collagen, phex, and sp7 (osterix) was evaluated at 4 and 8 weeks to determine bone formation across the defect. results: alp activity on the scaffold with ha demonstrated an approximate tenfold increase to that of the collagen scaffold alone. while a dose-dependent effect, with higher doses of exosomes resulting in a greater amount of alkaline phosphatase expression, expression that exceeded that of the 50ng bmp-4 control. dose escalation from 1.5, 2, and 4e9 resulted in similar increases in expression that was statistically greater with the combination of the fleece with the exosome component. bone formation in the mouse calvaium did not demonstrate gap closure at 4 or at 8 weeks, but did demonstrate enhanced osteoclastivity and robust bone remodelling at the margins of the defect. summary/conclusion: bone marrow-derived exosomes dried into an electrospun fibrillar collagen demonstrated in vitro osteoinductive potential that might provide site-specific placement that could enhance biologic potential. with the capacity for ambient temperature storage, the provision of site-specific placement becomes a technical consideration. placement of the human tissue derived exosomes in a transgenic mouse calvarial defect model did not demonstrate bridging bone across the defect. exosomes loaded with pten-interfering rna enables functional recovery in rats after complete spinal cord transection daniel offen a , nisim perets a , shaowei guo b , oshra betzer c , rachela popovtzer c and shulamit levenberg b a tel aviv university, tel aviv, israel; b technion, haifa, israel, haifa, israel; c bar ilan university, israel, ramt gan, israel introduction: complete spinal cord transection is a debilitating disease that usually leads to permanent functional impairments, with various complications and limited spontaneous recovery. the current investigation of molecular mechanisms controlling axon regeneration, (e.g., signalling networks and environmental cues), led to new strategies to enhance axonal regeneration. we have previously shown that intranasal administration of mesenchymal stem cells derived exosomes (msc-exo), cross the blood-brain barrier and significantly ameliorate motor and behavioural phenotype in several animal models of neurotrauma and neuropsychiatric disorders. methods: msc-exo were isolated from human bone marrow and were loaded with phosphatase and tensin homolog small interfering rna (pten-sirna). the exosomes were given intranasally to rats two hours after complete spinal cord transaction. eight weeks later we followed the motor function and histology and electrophysiology study was performed in order to reveal the connectivity and the biochemical changes in the treated rats. results: we demonstrate that intranasal (in) administrations of msc-derived exosomes could penetrate the blood-brain barrier, home selectively to spinal cord lesion via chemotaxis, and integrated in neurons within the lesion. furthermore, in rats with complete spinal cord transection, msc-exo loaded with pten-sirna silenced pten protein expression in the lesion and promoted robust axonal regeneration and angiogenesis, companied with decreased astrogliosis and microgliosis. moreover, the intranasal treatment partially restored electrophysiological and structural integrity, and most importantly, enabled the remarkable functional recovery and significant improvement in their movements. summary/conclusion: this rapid, non-invasive, approach, using cell-free nano-swimmers carrying molecules to target pathophysiological mechanisms suggest novel strategy for clinical translation to spinal cord injury and beyond. a novel umbilical cord derived wharton's jelly formulation for regenerative medicine applications introduction: musculoskeletal injuries have traditionally been treated with activity-modification, physical therapy, pharmacological agents and surgical procedures. these modalities have limitations, as well as potential side-effects. over the last decade, there has been an increased interest in the use of biologics for regenerative medicine applications (rma), including umbilical cord (uc) derived wharton's jelly (wj). despite this increase, there is insufficient literature assessing the amount of growth factors, cytokines, hyaluronic acid (ha) and extracellular vesicles (ev) including exosomes in these products. the purpose of this study was to develop a novel wj formulation and evaluate the presence of growth factors, cytokines, ha and ev including exosomes. methods: wj was isolated from human-uc obtained from consenting c-section donors and formulated into an injectable form. randomly selected samples from different batches were analysed for sterility testing and quantified for presence of growth factors, cytokines, ha and particles in ev size range. the results showed all samples passed the sterility test. growth factors including igfbp 1, 2, 3, 4 and 6, tgfα, pdgf-aa were detected. expression of several immunomodulatory cytokines, rantes, il-6 r, il-16, were also detected. expression of pro-inflammatory cytokines mcsfr, mip-1a; anti-inflammatory cytokines tnf-ri, tnf-rii, il-1 ra; and homoeostatic cytokines timp-1 and timp-2 were observed. cytokines associated with wound-healing, icam-1, g-csf, gdf-15, and regenerative properties, gh were also expressed. high concentrations of ha were observed. particles in the ev size range (30-150 nm) were detected and were enclosed by the membrane, indicative of true ev. summary/conclusion: our results confirmed the presence of numerous growth factors, cytokines, ha and ev in the wj formulation. more studies are underway to confirm the presence of exosomes in detected ev using exosome-specific markers. we believe the presence of multiple factors within one wj formulation may play a role in reducing inflammation, pain and augment healing of musculoskeletal injuries. this offers a potential expanded use for rma. funding: this study was funded by biointegrate llc, new york, ny, usa. collagen sponge loaded with mesenchymal stem cell-derived small extracellular vesicles promote robust bone regeneration shang jiunn chuah a , chee weng yong a , jacob ren jie chew a , ruenn chai lai b , yi ann cheow a , raymond chung wen wong a , asher ah tong lim a , sai kiang lim c and wei seong toh d introduction: mesenchymal stem cell (msc) therapy has demonstrated effective bone regeneration in clinical studies. however, the therapeutic efficacy of mscs have been attributed to the secretion of extracellular vesicles (evs), particularly 50-200 nm small evs (sevs). here, we investigate the efficacy of msc-sevs loaded in collagen sponge in the regeneration of critical-sized calvarial defects in immunocompetent rats. methods: sevs were isolated from conditioned medium of human mscs and stored at −20 c. calvarial defects of 8-mm diameter were surgically created on thirty-two 10-week-old male sprague-dawley rats. these rats were then randomly assigned to 2 groups (n = 16 rats/group): defects treated with collagen sponge containing 100 μg of sevs in 100 μl saline (cs/sevs) and defects treated with control collagen sponge containing an equivalent volume of saline (cs/control). at 1 and 8-week post-surgery, the calvarial bone samples was harvested for analyses by micro-computed tomography (micro-ct), histology, immunohistochemistry and histomorphometry. results: at 1-week post-surgery, micro-ct analysis showed little bone formation at the defect site in both cs/sevs and cs/control groups. no statistical differences were observed in micro-ct and histology scores in both groups. interestingly, cs/sevs group showed significantly higher osteocalcin (ocn)+ area of 5.7 ± 2.4% than that of cs/control group (2.1 ± 1.3%; p = 0.003). cd31+ microvessels at sizes ≤50 µm and >50 µm in cs/sevs group (34.4 ± 2.9 and 8.6 ± 2.1 microvessels/hpf) were also significantly higher than that of cs/control (18.8 ± 1.1 and 5.2 ± 1.4 microvessels/hpf; p = 0.002 and p = 0.038 respectively). by 8 weeks, cs/sevs group displayed enhanced new bone formation that completely bridged the calvaria defect. in contrast, rats in cs/control showed limited bone formation. consequently, cs/ sevs group displayed a micro-ct score of 3.9 ± 0.2 which was significantly better than that of cs/control group (2.5 ± 0.8; p = 0.001). cs/sevs group also exhibited >twofold increase in bone volume, and improved bone quality with higher trabecular thickness and number, and smaller separation (p < 0.01), compared to cs/control group. consistently, cs/sevs group displayed a significantly better histology score of 5.4 ± 1.0 than that of cs/control (2.6 ± 1.8; p = 0.001). moreover, cs/sevs group showed significantly higher ocn+ area of 48.9 ± 7.9% than that of cs/control group (20.4 ± 4.4%; p = 0.004). summary/conclusion: this study demonstrates that single-stage implantation of collagen sponge loaded with ready-to-use msc sevs can promote robust bone regeneration in a rat calvarial defect model. funding: national university of singapore, r221000114114, national medical research council singapore, r221000123213. immunomodulatory potential of extracellular vesicles derived from mesenchymal stromal cells introduction: extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) are promising new agents in regenerative medicine and immunotherapy. considering that independent msc-ev preparations might differ in their therapeutic function, we have set up a functional assay allowing testing for the potential immunomodulatory properties of independent msc-ev preparations. methods: human peripheral blood-derived mononuclear cells (pbmcs) were pooled from up to 12 different healthy donors warranting high allogeneic cross-reactivity, even following an optimized freezing and thawing procedure. after thawing, mixed pbmcs were cultured for 5 days in the absence or presence of msc-evs. thereafter, cell morphologies were documented, supernatants were harvested for cytokines quantification and cells were phenotypically characterized by flow cytometry. by analysing the expression of a collection of different lineage and activation markers, we selected a panel of antigens apparently being regulated by msc-ev preparations considered to be therapeutically active. results: we observed that in the presence of active msc-ev preparations more cd14+ (monocytes) are recovered from the mlr assay than in corresponding control samples. focusing on t cells, we learned that active msc-ev preparations reduced the content of cd4 and cd8 t cells expressing activation markers like cd54 and cd25. summary/conclusion: the mlr assay allows elaborated functional testing of immunomodulatory activities of given msc-ev preparations. currently, we are comparing the immune modulatory capabilities of evs derived from distinct sources and optimize the marker panel to distinguish discrete immune cell subtypes such as different cd4 cell types, i.e. th1, th2, th17 and tregs. extracellular vesicles in platelet-rich plasma: dependency on sample processing zala jan a , saba battelino b , darja božič c , matej hočevar d , ales iglič e , marko jeran c , manca pajnič a , ljubiša pađen a , domen vozel f and veronika kralj-iglič a introduction: platelet-rich plasma (prp) proved effective in regenerative medicine. numerous protocols for its preparation and application are available in the published literature. prp possesses important immune, haemostasis and regenerative factors, however, the mechanisms of their action are yet poorly understood. extracellular vesicles (evs) could be one of the important factors that would contribute to the beneficial effects of preparations. this study was performed as a part of a registered randomised controlled clinical trial (nr: nct04281901). prp was used to treat chronic middle ear inflammations. here we present the results of prp analyses from 29 blood samples of volunteers with no record of disease. methods: plasma obtained from 20 ml of blood was depleted of erythrocytes and enriched with other particles by repetitive centrifugation of samples. flow cytometry (fcm) was employed to monitor particle contents (cells and smaller particles) throughout the sample processing. the platelet gate was divided into two parts: intact platelets and smaller particles. identity and morphology of particles in the preparations were examined by scanning electron microscopy (sem). standard laboratory tests of blood were performed. results: sem images revealed the presence of heterogeneous population of particles in the preparation of prp, most of which were activated and partially fragmented platelets. the population of smaller particles measured with fcm, was identified as evs. the erythrocyte sedimentation rate was statistically significantly correlated to the volume of plasma obtained in the initial centrifugation step (r = 0,70, p < 0,001) and to the concentration of evs (r = 0,82; p < 0,0001). time from sample collection to the preparation of prp was negatively correlated with the concentration of platelets in prp and positively with the concentration of evs (r = 0,75, p < 0,001). platelet concentration in preparation samples was found to depend on the concentration of platelets in the blood and parameters of sample processing connected with larger centrifugal and shear forces on the samples during centrifugation. these include: sample volume, the size and shape of the centrifuge tube and the distance of the sample from the rotor axis. summary/conclusion: evs are gradually forming upon activation and degradation of cells in the sample throughout the sample processing. optimal processing may importantly contribute to the healing properties of preparation. funding: authors acknowledge support from the european union's horizon 2020 research and innovation program under grant agreement no. 801338 (ves4us project) and slovenian research agency (arrs, grants p2-0232, p3-0388, j1-9162). satellite cell-derived extracellular vesicles as a therapeutic for mitochondrial dysfunction in duchenne muscular dystrophy duchenne muscular dystrophy (dmd). sc-derived extracellular vesicles (sc-evs) may unlock the therapeutic potential of scs by overcoming these limitations. to investigate their therapeutic potential, we assessed the ability of sc-evs to reverse mitochondrial dysfunction, a key pathological feature of dmd, in oxidatively-damaged c2c12 and primary dmd myotubes. methods: scs from c57 mice were isolated and cultured. evs were isolated from the supernatant of scs via polyethylene glycol precipitation and characterized using nanoparticle tracking analysis. the ability of sc-evs to deliver protein cargo to c2c12 myotubes, and the localization of the cargo once delivered, were analysed using fluorescence microscopy. to examine sc-ev potential to restore the function of damaged mitochondria, c2c12 myotubes were treated with 50 µm h2o2 for 24 h followed by treatment with 3.12x108 sc-evs for 24 h. separately, cultured dystrophic myotubes were treated with 3.12 × 108 evs every 24 h for 72 h. in both sets of experiments, maximal oxygen consumption rate (max ocr) was measured via seahorse xf cell mito stress test. where appropriate, a t-test was performed to test for statistical significance (p < 0.05). results: based on estimated cell number and ev quantification, each sc released approximately 2.35 × 105 ± 3.10x104 evs/day. evs delivered protein cargo into myotubes within 2 h. fluorescent labelling of intracellular mitochondria showed co-localization of delivered protein and mitochondria. incubation of myotubes with h2o2 resulted in a 42% decline in max ocr relative to untreated myotubes. subsequent treatment with sc-evs resulted in a 76% increase in max ocr. treatment of undamaged myotubes with sc-evs had no effect on max ocr. primary dmd myotubes treated with sc-evs showed a 78% increase in max ocr relative to untreated dmd myotubes. summary/conclusion: sc-evs rapidly deliver proteins into myotubes, much of which co-localizes with mitochondria, and reverses mitochondria dysfunction in oxidatively-damaged and dystrophic myotubes. introduction: flow cytometry has been used extensively for analysis of ev particles stained with fluorescent antibodies directed to the known cell surface markers. quantitation of the surface markers in terms of the number of molecules or the number of antibodies bound per specific marker has remained one of the largest challenges in the ev research field. changes in instrument setup as well as changes in fluorescent antibodies from different vendors, all impact the relative mfi values for the same ev sample. in this work we report a standardization method of quantitating extra-cellular vesicle surface markers with mesf liposomes. methods: liposomes labelled with fitc fluorescent dye were prepared with a bd proprietary technology. dynamic light scattering analysis was used for size determination of the liposomes. bd facsaria™ fusion system, modified with a small particle side scatter module (sp ssc), was used for analysis of the labelled liposomes by flow cytometry. results: we created a set of 180 nm fitc-modified liposomes of various fluorescent intensities with a known number of fitc molecules incorporated in each liposome intensity. the mfi values of each liposome population (intensity) had a linear relationship to the amount of fitc used for labelling the liposome nanoparticles, suggesting that no self-quenching of fitc fluorescence had occurred. the number for the fitc fluorophores for each liposome intensity was expressed in the units of molecules of equivalents soluble fluorochrome (mesf). a plot of mesf vs. the fluorescent intensity of the liposomes (mfi values) obtained from flow cytometry analysis provided a calibration curve, from which the fluorescent intensity (mfi value) of a stained ev sample can be converted to the number of fluorophores bound (mesf value) to the surface of the ev particles. summary/conclusion: by this approach, the mfi values of stained ev particles are converted to standardized mesf values that are independent of instrument variation, resulting in further improvement of inter-laboratory standardization. furthermore, utilization of liposomes with similar size and refractive index to ev particles simplifies the data evaluation and improves the accuracy of ev surface marker quantitation by flow cytometry. currently, other fluorescent dyes are being explored to expand the utility of mesf liposomes with other fluorescent colours. measuring cholesterol as a high-throughput method for quantifying extracellular vesicles introduction: the extracellular vesicle (ev) field currently lacks a high-throughput method for accurately quantifying evs in solution. ev quantification has traditionally relied on nanoparticle tracking analysis (nta), which is time intensive and indiscriminately counts non-ev particles, such as membrane fragments and protein aggregates. we have rigorously assessed two commercially available methods for measuring cholesterol, a major lipid component of the ev lipid bilayer, and evaluated the utility of these assays to quantify evs in minimally processed samples. methods: the amplex® red cholesterol assay and cedex bio ht were used to quantify cholesterol in ev samples via enzymatic oxidation, with dynamic ranges of 80-8,000 ng/ml and 4-800 µl/ml, respectively. samples throughout various stages of purification were analysed, from clarified cell culture medium to highly purified evs separated on an iodixanol gradient. we evaluated several pre-processing methods, to remove non-ev cholesterol content prior to analysis. results: the amplex® and cedex bio ht assays were found to perform comparably for quantifying cholesterol in purified evs (r2 = 0.92). importantly, cholesterol quantification on purified ev samples, ranging from 1e11 to 4e13 particles/ml, correlated well with nta measurements (r2 = 0.96). both 45 µm filtration or an additional 16,000 rcf centrifugation step following clarification removed cholesterol associated with cellular debris or other non-ev sources, allowing for accurate quantification of conditioned medium samples or ultracentrifugation pellets (ucp) instead of needing to rigorously purify samples with an iodixanol density gradient. summary/conclusion: cholesterol quantitation can be used to accurately estimate ev concentration, allowing for rapid characterization of samples from clarified cell culture supernatant to highly purified evs. this highthroughput analytical capability may enable more comprehensive assessment of methods to boost ev yield through mass screening of cell culture conditions. optimization of nanoparticle tracking analysis of extracellular vesicles isolated from plasma and bronchopulmonary lavage fluid of patients with non-small cell lung cancer introduction: recent studies show that tumourderived extracellular vesicles (evs) greatly influence the tumour microenvironment and impact the therapy. in non-small cell lung cancer (nsclc), bronchopulmonary lavage fluid (balf) appears to be a good source of tumour-derived evs, providing more accurate information about the tumour microenvironment than evs from plasma. so far there is a lack of accurate and standardized methods for ev quantification. fluorescence nanoparticle tracking analysis (fl-nta) is an emerging method of ev-analysis, allowing discrimination of evs and exosomes from impurities. here we perform an optimization of the fl-nta method to compare evs from plasma and balf of nsclc patients and healthy controls (nc). methods: evs were isolated using homemade sizeexclusion chromatography (sec) columns (plasma) and ultrafiltration or differential ultracentrifugation (balf). nta was performed using zetaview pmx220 (particle metrix) after ev-staining with membrane dyes or fluorescence-labelled antibodies against typical ev-marker (cd9, cd81, cd63). results: nta scatter measurements showed a higher total particle concentration in plasma than in balf. however, membrane-specific staining showed a much greater purity of ev-preparations from balf, where nearly 100% of the particles detected in scatter mode showed positive membrane-staining. in contrast, only around 40-50% of particles in the plasma ev-preparations were positive for the membrane dyes. fluorescence-staining for ev surface marker requires further optimization to obtain reproducible results. summary/conclusion: classical nta using only the scatter mode fails to discriminate between evs, lipoproteins and protein aggregates. for ev-analysis from complex biofluids like plasma, fla-nta and staining for specific ev marker is necessary to receive reliable data. balf seems to be a better source of tumourderived evs than plasma, since the obtained ev-preparations show a higher purity. improving conditions for fluorescence-staining and nta measurement of evs from plasma and balf of nsclc patients will provide an additional method for quantifying and phenotyping of evs. introduction: the exoviewer platform currently enables the user to capture extracellular vesicles (ev) by means of surface antigen-specific antibodies (e.g. targeting tetraspanins), making possible the enumeration of individual particles using single-particle interferometric reflectance imaging sensor (sp-iris, interferometric) imaging as well as fluorescence. currently, through interferometric imaging particles smaller than 50 nm cannot be detected, while fluorescently stained ev smaller than 50 nm can be well resolved. further, it is conceivable that small ev contain antigen numbers in the single digits, making antigen-specific immunostaining a challenge. to further characterize ev populations of different sizes and surface marker composition, it would be highly advantageous to target the vesicular nature of the detected particles linked to a fluorescence readout. methods: the goal of this project is to detect ev with a probe that is ubiquitously distributed across the surface (or lumen) of the vesicle. small (30-150 nm) ev present fairly distinctive lipid membrane features in the extracellular environment, turning the ev membrane into a "universal" marker, and as such may serve as an alternative marker that is complementary to canonical ev surface markers. results: here we present data on successfully staining ev with the membrane dye di-8-anepps (di-8) and the luminal dye calcein-am. we demonstrate that ev from different sources can be efficiently stained with either dye, allowing the quantitative characterization of ev in an unbiased manner using exoviewer's fluorescence mode. while both dyes certainly have their own unique strengths, they exhibit the wanted linear correlation of ev staining versus concentration. further, both dyes are compatible with subsequent immunostaining applications, allowing the user to target specific surface or luminal markers (di-8). summary/conclusion: while a large-panel screening featuring other powerful dyes is continuously ongoing, the current data support the notion of providing the experimenter with a reference for total particle count and at the same time fully exploring the larger dynamic range of the fluorescence mode. moreover, the universal probe will enable the user to correlate intensity and particle size measurements, thereby significantly improving the exoviewer platform and its applications. membrane labelling is essential for the identification and quantification of extracellular vesicles via facs introduction: extracellular vesicle (ev) research is challenged by the lack of standard protocols to identify and distinguish between exosomes and ectosomes being released via exocytosis or plasma membrane shedding, respectively. analysis of small ev populations requires high-resolution technology and can be further improved using fluorescent labels such as carboxyfluorescein diacetate succinimidyl ester (cfse). at the inner leaflet of the plasma membrane, cfse is cleaved enzymatically resulting in covalent binding of the dye. in this study we optimized the conditions for membrane labelling of evs and their subsequent detection by flow cytometry to obtain a maximum yield of intact evs. methods: using sequential centrifugation, we separated ev subpopulations from supernatants of colo 357 pancreas carcinoma cells based on size and mass. after 10,000x g centrifugation, we reconstituted evs from the pellet. we used cfse for ev detection and analysed the expression of tetraspanins by facs to confirm the lipid bilayer structure. furthermore, we determined size distribution of evs by nanoparticle tracking analysis (nta) and electron microscopy. detecting evs as cfse+ events, we quantified our samples and investigated the impact of threshold adjustment on ev quantification. results: after high speed centrifugation of cell free supernatants, we identified cfse+ events as evs, which appeared as round structures under the microscope, and ranged from 80 to 40 nm in size. interestingly, tetraspanin markers cd9 and cd81 were detectable only on a subpopulation of purified evs, suggesting heterogeneity of our preparations. for sufficient labelling of evs, minimal temperature variations and short incubation times correlated with ev stability. of note, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of labelled evs and hence, is central for data comparability. summary/conclusion: protocol standardization is of major importance for the use of evs as diagnostic markers in liquid biopsies. funding: this project has been supported in part by annelise-asmussen foundation, luebeck (grant 180802), leo pharma germany (grant 180208). surface plasmon field-enhanced fluorescence spectroscopy (spfs) system for quantitative and qualitative extracellular vesicles total evaluation without any sample pretreatment introduction: the function of extracellular vesicle (ev) is interested in the immunology and oncology fields as a key transmitter for cellular communication. however, the conventional ev evaluation methods are required complicated evs preconcentration from the sample, its leads ev analysis uncertainty. in this study, we applied the spfs highly sensitive automated system for quantitative and qualitative ev evaluation without any sample pre-concentration and preparation step. methods: spfs automated system and plastic disposable sensor had been developed by konica minolta corporation in house. anti-membrane protein (cd9, cd63, cd83) antibody was chemically bonded on hydrophilic polymer which was immobilized through the gold thin film on the spfs sensor. the concentration of standard ev materials was evaluated by the qnano system before using. ev detection without preconcentrating was achieved by sandwich immunoassay step in microchannel round-trip flow reaction (tat 120 min) with the spfs system, and elisa was adapted as a conventional standard method. after spfs highly sensitive fluorescent measurements step, extracted and detected ev were effectively recovered by using the recovery buffer reaction. results: the ev sensitivity performance between spfs and elisa clearly showed a significant difference, and the lod of spfs (8.3 particles/μl) method was estimated 3000 times superior to the lod of conventional elisa (26,000 particles/μl). the spfs calibration curve showed a wide dynamic range at least over 5 logs as an additional specificity. spfs method also showed fine results in the dilution linearity test with high reproducibility under the serum/plasma sample condition. the data for recovery test of ev expected us that highly accurate measurement can be guaranteed under the condition of dilution about 10 times or less even in the whole blood sample. after the spfs measurement, extracted ev on the spfs sensor chip could be effectively recovered and could be analysed nucleic acid which contains micro rna. summary/conclusion: spfs system might have great potential for quantitative and qualitative ev evaluation. our strategy with spfs system for ev proteomic and genomic profiling will be possible for applying to ev quality control as well as a novel biomarker development. identification of a novel compound that inhibits small ev secretion and tumour progression by a sensitive elisa screening. yunfei ma a , takeshi yoshida a , duc tuan nguyen a , kazutaka matoba b , katsuhiko kida b , taito nishino b and rikinari hanayama c a kanazawa university, kanazawa, japan; b nissan chemical corporation, tokyo, japan; c wpi nano life science institute, kanazawa university, kanazawa, japan introduction: small evs from tumour cells are known to promote tumour progression, therefore, it is expected to develop drugs that regulate small ev secretion, which can be used in clinical applications. methods: to identify such regulators, we first developed a sensitive elisa system for the quantification of small ev secretion using a high-affinity ev binding protein tim4. by using this elisa system, we screened for small compounds that promote or inhibit small ev secretion using a drug-repositioning compound library (about 1,600 compounds). results: as a result, we identified eight promoters and two inhibitors, including compound a, which significantly reduced small ev secretion from various cell types without affecting cell growth. we further investigated the effects of compound a on a mouse model of osteosarcoma and found that compound a suppressed tumour progression efficiently. summary/conclusion: these data suggest that compound a would be useful not only for the characterization of small ev function but also for the clinical therapy against tumour progression, by inhibiting small ev secretion. introduction: for many years it was believed that several proteins such as cd63, cd9 and flotillin-1 were unique for exosomes, however recent studies have shown that several of these markers also can be present in other subpopulations of evs (kowal et al pnas 2016) . furthermore, few markers have been identified as uniquely present in microvesicles. the aim of this study was to in depth compare the proteome of microvesicles and exosomes. methods: mda-mb-231-luc-d3h1, -d3 h2ln and -bmd2a were cultured in ev-depleted media. microvesicles (16,500 x g, 20 min) and exosomes (118,000 x g 2.5 h) were isolated using a combination of differential ultracentrifugation and a density cushion (~1.1 g/ml). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, and electron microscopy (em). quantitative mass spectrometry (tmt-lc-ms/ms) was used to identify differently enriched proteins in microvesicles and exosomes (n = 3 x 3 cell lines). results: in total 6493 proteins were quantified, with 4851 being quantified in all samples. in total 818 and 1567 proteins were significantly upregulated in exosomes and microvesicles, respectively. go terms associated with the proteins significantly upregulated in exosomes were "extracellular exosome" and "plasma membrane", while the microvesicle proteome was associated with "membrane" and mitochondrion". in exosomes tetraspanins, annexins, escrt and rab proteins were significantly upregulated. in contrast, proteins that were upregulated in microvesicles were involved in protein translocation into the mitochondrial membrane (timm and tomm proteins), in cytokinesis, and in micos complex. however, flotillin-1 was not differently expressed in the ev subtypes. summary/conclusion: this study identifies several proteins to be differently enriched in exosomes and microvesicles. several of the proteins suggest recently by kowal and colleagues, such as adam10 and mitofilin could be validated. additionally several novel proteins could be identified. identifying markers separating microvesicles and exosomes is of high importance for the ev field and future studies will have to validate them also in other cells to determine if they are generic. introduction: the cellular elements composing the lining of brain ventricles have drawn much attention from neuroscientists, especially the role of subependymal cells in neurogenesis, but the role of ependymal cells in brain function and disease is still neglected. our objective is to study the morphological aspects of rat brain ventricles and the ependymal cells as analysed by transmission and field emission scanning microscopy in normal or ischaemic rats. methods: for this purpose, male wistar rats were submitted to 10 minutes of global brain ischaemia and divided into two groups: a) sham-operated animals and b) saline-treated ischaemic animals. all animals were allowed to survive for seven days. all procedures were approved by the ethics committee of the federal university of são paulo (2018/7633081117). transmission and scanning electron microscopic analysis of lateral brain ventricles were done in buffered 2,5% glutaraldehyde/2%formaldehyde perfused brains. cerebrospinal fluid was collected for nta analysis. results: the morphological characterization of brain ventricle revealed a slight rarefaction of ciliary tufts of animals submitted to ischaemia when compared to normal animals. field emission electron microscopy revealed the secretion of vesicles by the ependymal cilia in the lateral ventricle. size and concentration of particles in the cerebrospinal fluid was confirmed by nta and transmission electron microscopy. summary/conclusion: our results are unprecedented and bring innovative potential regarding the role of extracellular vesicles in both the physiology and pathogenesis of the nervous system. these data may also contribute to the development of new technologies for diagnosis and therapy of chronic degenerative diseases. introduction: the function of mitochondria relies on precise and effective quality controls. neurons have high metabolic demands and employ multiple mechanisms to ensure functional mitochondria. we investigated mitochondrial vesiclesa less understood quality control mechanism for mitochondriaand assessed the effect of cellular stress. methods: we surveyed mitochondrial vesicles in rat and planaria brains with electron microscopy. we quantified these vesicles with serial-section electron microscopy (fib-sem). we also conducted confocal microscopy with airyscan analysis of cultured neurons expressing fluorescently tagged mitochondrial markers. results: electron microscopy showed the ultrastructure of various types of mitochondrial vesicles. serial-section electron microscopy revealed the 3d ultrastructure of mitochondrial vesicles and their prevalence in neurons. confocal microscopic analysis showed increased numbers of mitochondrial vesicles in neurons under mild stress. summary/conclusion: our findings provide direct structural evidence for mitochondrial vesicles in neurons and their abundance in response to neuronal stress. their detection in the extracellular compartment (evidence for which is expected to be presented by the time of isev) may allow for development of biomarkers for mitochondrial health, with relevance to numerous pathologic conditions. from endosomes, might be involved in the impairment of rna, specific feature of als disease. combining high-resolution flow cytometry and surface marker analysis using an automated platform to study extracellular vesicle in cerebrospinal fluid unity health toronto, toronto, canada introduction: there is growing enthusiasm that extracellular vesicles (evs) carry the potential for a variety of applications in medicine. as biomarkers, evs may aid clinicians in the evaluation of diagnoses, disease progression, or even response to therapy. however, proper characterization of the amount, size, and phenotype of evs in a given sample remains challenging due to their sub-micrometre size and heterogeneity. over the last years, technologies, including high-sensitivity flow cytometry and automated platforms that simultaneously assess ev amount, size, and phenotype, have matured, providing new opportunities to study evs for future clinical applications. using such technologies to analyse cerebrospinal fluid (csf), which is in direct contact with the brain and spinal cord, may yield valuable insights into neurological disease processes. while there is often uncertainty about the exact source of evs in a biological sample, cd171 has emerged as a surface marker that suggests a neuronal origin. methods: csf samples that had been stored at -80 degrees celsius for advanced biomarker studies were analysed using two distinct approaches. a becton, dickinson and company (bd) aria iii flow cytometer was converted into using violet side scatter (ssc) for improved detection of evs with 405 instead of 488 nm ssc. for the combined analysis of amount, size, and phenotype, samples were analysed with the nanoview bio r100 platform. phenotype analysis included probing for the classic tetraspanins associated with exosomes (cd9, cd63, cd81) and the neural cell adhesion molecule l1 (cd171). results: flow of csf samples showed similar vesicle counts in control vs. disease and an increase of counts in later disease stages when neurodegeneration is thought to be more prominent. all csf samples showed some binding to classic exosomal markers (cd9, cd63, cd81). the sample taken at the latest time point showed relatively high vesicle counts, overall larger vesicle size, and abundant cd171 binding. interestingly, the cd171 positive evs were not positive for any of the classic exosomal markers (cd9, cd63, and cd81). summary/conclusion: this data supports the notion that analysing the amount, size, and surface markers of evs in csf can reveal intriguing dynamics in such basic ev characteristics over time and suggests important differences between ev populations in different disease stages. while previous studies indicated that cd171 could identify an ev to be of neuronal origin, it remains to be determined whether such specific surface markers will emerge as clinically relevant tools to support the evaluation of people affected by neurological diseases. a distinct microrna signature in plasma derived small extracellular vesicles of different neurodegenerative diseases introduction: exploring identifying robust biomarkers is essential for early diagnosis of neurodegenerative diseases. blood stream transports large (levs) and small extracellular vesicles (sevs), which are extracellular vesicles of different sizes and biological functions that are transported in blood. aim of our study was to investigate mrna/mirna signatures in plasma derived levs and sevs of amyotrophic lateral sclerosis (als), alzheimer's disease (ad), parkinson's disease (pdpd), fronto-temporal dementia (ftd) and alzheimer's disease (ad) patients. methods: levs and sevs were isolated from plasma of patients and healthy volunteers (ctr) by ultracentrifugation and rna was extracted. whole transcriptome and mirna libraries were prepared with truseq stranded total rna kit and truseq small rna library kit (illumina). results: our data suggested that the rna cargo in levs and sevs varies among different diseases. mirna analysis in sevs provided the most informative disease specific signatures, while whole transcriptome analysis did not show any specific signature. als was characterized by a small but specific group of circulating mirnas. mirnas profiling revealed that pd and ftd can be subgrouped in two classes while ad appears to be a homogeneous disease population. furthermore, mirnas profiling show the presence of overlaps in the signatures between the analysed diseases. mirna profiling in levs is similar to that observed in sevs, although in levs the overall differences between diseases are less marked. summary/conclusion: in this study we have demonstrated that mirnas are the most interesting subpopulation of transcripts transported by plasma derived sevs since they discriminate a disease from the other and they can provide a signature for each neurodegenerative diseases. may be linked with apoe genotype, we investigated the possible effect of apoe genotype on brain-derived evs (bdevs) and their protein and rna molecular cargo. methods: cortical brain tissues of ad patients with different apoe genotypes [ε2/ε3 (n = 5), ε3/ε3 (5), ε3/ε4 (6), ε4/ε4 (6)] and non-ad controls (n = 7) were obtained. bdevs were separated by size exclusion chromatography plus ultracentrifugation (uc) and characterized per misev2018. proteins were analysed by mass spectrometry. after protein identification, data were normalized using the cyclicloess method and analysed by principal component analysis (pca). nested factorial design highlighted differentially expressed proteins. rna from bdevs was extracted by mirneasy mini kit. small rna libraries were constructed using the ion total rna-seq kit and sequenced on the ion torrent s5™ using ion™ 540 chips. reads were aligned to human reference transcriptomes using bowtie. differential gene expression was quantified by edger and limma. results: among 28 proteins dysregulated in ad bd-sevs, several have reported roles in ad, e.g., microtubule-associated protein tau and peroxiredoxin-6. regarding apoe genotypes, 16 proteins were differentially expressed between ε4 carriers (ε3/ε4 and ε4/ε4) with non ε4 carriers (ε2/ε3 and ε3/ε3). however, ev markers did not differ by apoe genotype. in contrast to protein cargo of bdevs, the overall small rna expression pattern was similar among ad patients with different apoe alleles and non-ad patients. only a few mirnas showed different abundance level between ε2/ε3 and ε4/ε4 groups, or between ad and non-ad groups. summary/conclusion: bdevs carry proteins and mirnas related to ad development and apoe genotypes. further verification of protein and rna expression in brain and plasma derived evs may reveal mechanisms of ev function in neuroinflammation and develop biomarkers for ad disease. funding: this project was funded by mh118164. efficient pathology spread by extracellular vesicles from human brain tissues in mouse brain and tissue cultured neurons: transmission and propagation to gabaergic neurons however, whether human brain-derived evs induce tau pathology has not yet been characterized in the mouse brain. here, we assess the mechanisms of disease spread after intrahippocampal injection of human brainderived evs into the aged mouse model. methods: ev-enriched fractions were isolated from unfixed frozen human brain samples from ad, prodromal ad (pad), control (ctrl) cases, and tau knockout (tko) mouse brains. isolated evs containing 300 pg of human total tau were sterotaxically injected into the right outer molecular layer of the dentate gyrus of 18 months-old c57bl/6 female mice. 4.5 months after the injection, hippocampal slices were prepared for whole-cell patch clamp recordings of ca1 pyramidal neurons were undertakent. hippocampi were analysed with immunohistochemistry using phosphorylated-tau (p-tau) epitopes including at8. evs were examined for protein composition by protein mass-spectroscopy, the neuronal uptake in vitro, and structural analysis by the atomic force microscopy (afm). results: semiquantitative brain-wide immunohistochemistry of p-tau revealed that inoculation of ad or pad-evs induced tau propagation throughout the hippocampus, including the dentate gyrus, ca3 and ca1 subregions. at8 was localized primarily in gad67 + gabaergic neurons in pad and ad evs groups, accompanied with reduced amplitude of inhibitory postsynaptic currents and excitatory-inhibitory ratio in amplitube of postsynaptic currents in ca1 pyramidal neurons in pad evs. afm analysis showed higher density of tau oligomers in both ad and pad evs while only ad evs showed significantly higher neuronal uptake compared to ctrl evs. finally, proteomic analysis showed that ad evs are enriched in disease and glia-related molecules compared to ctrl evs, which may contribute to their enhanced neuronal uptake. summary/conclusion: intracranial injection of ad or pad evs induced p-tau accumulation primarily in gabaergic neurons throughout the hippocampus, resulted in higher uptake by neurons, and tau oligomer conformation, indicating of their pathogenic potency as seeding factors. gabaergic neuronal dysfunction in the hippocampal neuronal circuitry reported in early ad brains could be attributed to specific ev mediated tau propagation in this cell type, a phenomenon meriting further investigation and validation. funding: nih rf1ag054199, nih r01ag054672, nih r56ag057469, cure alzheimer's fund, brightfocus foundation, curepsp, coins for alzheimer's research trust introduction: extracellular vesicles (evs) are released by cells of the central nervous system as a result of injury, including mild traumatic brain injury (mtbi). since mtbi may alter circulating levels of evs, this study aimed to investigate differences in circulating ev numbers between contact sport athletes with and without acute mtbi. methods: circulating evs containing cd63 (cd63 + ev), cd81 (cd81+ ev), and neural cell adhesion molecule (l1cam+ev) were analysed in young, male athletes with or without mtbi (18-29 yo, n = 6 per group). sodium citrate-treated blood samples were obtained from athletes with mtbi within 48-hours of injury and from control athletes free of mtbi for one year. athletes were best matched for age and history of prior mtbi. samples were double-centrifuged to obtain platelet-poor plasma and stored at −80°c until analysed. quantification of evs was performed using a spectral flow cytometer. the study was approved by temple university's irb, and all athletes provided written informed consent. results: mann-whitney u tests showed that population percentages of small size (179-304 nm) cd63 + ev, cd81+ ev and l1cam+evs were significantly higher in mtbi athletes (mean rank: 8.0, 9.5, 9. 3) than controls (mean rank: 3.6, 3.5, 3.7) (u = 3.0, p = 0.03; u = 0.0, p > 0.01; u = 1.0, p > 0.01, respectively). population percentages of large size (500-800 nm) cd63+ ev, cd81+ ev and l1cam+evs were also significantly higher in mtbi athletes (mean rank: 8. 2, 9.2, 9.5) than controls (mean rank: 3.4, 3.8, 3.5) (u = 2.0, p = 0.02; u = 2.0, p > 0.01; u = 0.0, p > 0.01, respectively). there were no significant differences between percentages of evs associated with blood brain barrier function (cd144+ ev) or platelets (cd42a+ev) among mtbi athletes or controls. introduction: parkinson's disease (pd) is characterized by clinical heterogeneity, different rates of progression and absence of definitive biomarkers. extracellular vesicles (evs) are easily isolated from plasma and play a central role in intercellular communication which is highly relevant for inflammatory processes implicated in protein misfolding-related neurodegenerative disorders. thus, we characterized distinctive plasmatic ev subpopulations of pd and atypical parkinsonisms (ap) patients, with the aim to identify candidate biomarkers among evs surface membraneproteins. methods: plasmatic evs were collected from 27 pd, 19 matched healthy controls (hc), 9 ap with multiple system atrophy (msa) and 9 ap with tauopathies (ap-tau). evs were quantified by nanoparticle tracking analysis. the expression of 37 ev-surface markers, related to inflammatory and immune cells, were measured by macsplex and correlated to clinical scales. a diagnostic model based on ev markers expression was built via supervised machine learning algorithms and validated in an external cohort (10 pd, 20 hc, 5 msa, 5 ap-tau). the cantonal ethics committee approved the study protocol. all enrolled subjects gave written informed consent. results: pd showed the highest ev concentration compared to others groups. pd and msa displayed a greater pool of overexpressed immune markers compared to ap-tau. ev antigens correlate to cognitive impairment and disease gravity in pd and msa. the roc curve analysis of a compound ev marker showed optimal diagnostic performance for pd (auc 0.908; sensitivity 96.3%, specificity 78.9%) and msa (auc 0.974; sensi-tivity100%,specificity94.7%)andgoodaccuracyforap-tau (auc 0.718; sensitivity 77.8%, specificity 89.5%). a diagnostic model based on ev markers expression, cor-rectlyclassified88.9%ofpatientswithreliablediagnostic performance after validation in an external cohort (77% of accuracy). summary/conclusion: this analysis of multiple immune surface markers of circulating evs in pd and ap well captured the clinical heterogeneity of pd and showed optimal diagnostic performance. furtherly it suggests a different immune dysregulation in pd and msa vs. ap-tau, to be confirmed by functional analysis in experimental models of disease. funding: supported by abreoc. separation and characterization of extracellular vesicles from human cerebrospinal fluid introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off 3,000), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions (3 + 4) after the sec void volume as evaluated by detection of cd63 and cd9 markers (immunoblotting) and annexin a2 (peptide mapping by nanolc-ms/ms). there, nanoparticles around 150 nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between 55 and 165 nm, with average diameter 94 ± 31 nm. cd63 was visualized by immunocytochemistry at the surface of ev around 80 nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin-3 binding protein (lgals3bp or 90 k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between 15 and 60 nm; some of those nanoparticles had ring-like appearance. occasionally 90 k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of 90 k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from 90 k nanoparticles to investigate their individual physiological relevance and biomarker potential. introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off 3,000), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions (3 + 4) after the sec void volume as evaluated by detection of cd63 and cd9 markers (immunoblotting) and annexin a2 (peptide mapping by nanolc-ms/ms). there, nanoparticles around 150 nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between 55 and 165 nm, with average diameter 94 ± 31 nm. cd63 was visualized by immunocytochemistry at the surface of ev around 80 nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin-3 binding protein (lgals3bp or 90 k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between 15 and 60 nm; some of those nanoparticles had ring-like appearance. occasionally 90 k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of 90 k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from 90 k nanoparticles to investigate their individual physiological relevance and biomarker potential. release of extracellular vesicles from platelets requires platelet-platelet interaction aleksandra gąsecka a , naomi c. buntsma b , sytske talsma c , krzysztof j. filipiak d , rienk nieuwland e and edwin van der pol f introduction: arterial thrombosis is a major and global cause of human death and disability, but a biomarker for early-diagnosis of thrombosis is absent. platelet activation and aggregation are the first steps of plateletrich thrombus formation, but their relative contribution to platelet extracellular vesicles (pevs) release is unknown. methods: to study the relation between pev release and platelet interaction (aggregation), citrate-anticoagulated whole blood (wb) from healthy donors was diluted 2, 4, 8, 16 and 32-fold and activated by 30 μm thrombin-receptor activating peptide (trap). in addition, undiluted wb and 10-fold diluted wb, which totally blocked pev release, were activated with various trap concentrations. concentrations of pevs (cd61 + and cd61+, cd62p + >1000 nm) and activated platelets (cd61+, cd62p+ >1000 nm) were measured by flow cytometry (apogee a60-micro). platelet aggregation was assessed using impedance aggregometry. results: a 10-fold dilution of wb blocked both aggregation and the release of pevs. compared to baseline, activation of undiluted wb with trap increased the concentrations of cd61 + 2.2-fold and cd61+-cd62p + pevs 7.2-fold. the concentration of cd61+ (r2 = 0.71) and cd61+-cd62p+ (r2 = 0.78) pevs as well as platelet aggregation (r2 = 0.8) scaled inversely (reciprocal) with the dilution of wb. further, we found a linear correlation between the % of activated platelets and the concentration of cd61+ (r2 = 0.80) and cd61 +, cd62p+ (r2 = 0.64) pevs in undiluted wb, which was absent in 10-fold diluted blood (r2 < 0.05). summary/conclusion: the absence of aggregation and pev release upon platelet activation in 10-fold diluted blood shows that aggregation directly depends on the distance between platelets, which is confirmed by the reciprocal relationship between pev release and blood dilution. because pevs are only released when platelet activation is followed by aggregation, pevs are a potential early biomarker of thrombosis. funding: ag is supported by the national science centre, research programme preludium 2018/31/ n/nz7/02260. evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni 15924. age-dependent alteration in concentration and size distribution of extracellular vesicles in plasma of normotensive and hypertensive rats kosuke otani, muneyoshi okada and hideyuki yamawaki laboratory of veterinary pharmacology, school of veterinary medicine, kitasato university, towada, japan introduction: spontaneously hypertensive rats (shr) are the most widely used animal model of human essential hypertension. we previously reported that plasma small extracellular vesicles (sevs) in shr regulate systolic blood pressure, however, the mechanism has not been clarified. in the present study, we compared the concentration and size distribution of plasma evs (sevs and large evs) from young and aged normotensive wistar kyoto rats (wky) and shr. methods: heparin-anticoagulated plasma was collected from male wky and shr at 5~7-(young) and 15-(aged) week-old. large evs were isolated from the plasma by centrifugation (10000 x g). sevs were isolated by ultracentrifugation (164,071 x g) following precipitation with polyethylene-glycol. the concentration and size distribution of sevs and large evs were measured by a tunable resistive pulse sensing analysis. results: there was no significant difference in the total concentration of plasma sevs between wky and shr or between young and aged rats. the mean diameter of plasma sevs from aged rats was larger than that from young rats in both wky and shr. also, the number of particles with a diameter of smaller than 150 nm in plasma sevs from aged rats was lower than that from young rats. the concentration of plasma large evs from aged rats was higher than that from young rats in both wky and shr. there was no significant difference in the size distribution of plasma large evs between wky and shr or between young and aged rats. summary/conclusion: the present results for the first time demonstrate that the concentration of plasma large-sized evs is increased by ageing, while there is no difference in the concertation and size distribution of evs between wky and shr. further research is required to clarify the cause of age-dependent alternation in plasma ev size distribution and its physiological meaning. microrna profiling of circulating extracellular vesicles is involved with susceptibility to age-related diseases: relevance to cardiovascular signalling in ageing process ionara rodrigues siqueira a , laura cechinel b , rachael batabyal c and robert freishtat c a universidade federal do rio grande do sul (ufrgs), porto alegre, brazil; b universidade federal do rio grande do sul, porto alegre, brazil; c children's national hospital, washington, usa introduction: ageing represents a central risk factor for several diseases, such as cardiovascular diseases. our hypothesis is that extracellular vesicles (evs) can be potential mechanism of spreading molecules, such as micrornas, involved with susceptibility to chronic age-related diseases and geriatric syndromes. in this context, the role of micrornas in age-induced detrimental changes in the cardiovascular system has been suggested. although evs can protect micrornas from endogenous rnases and internalization of these vesicles into cells is involved with cell communication, delivering micrornas even to distant tissues, the relationships between evs micrornas profile and chronic age-related diseases has not been evaluated. our aim was to investigate the microrna profile of circulating evs during ageing process and their downstream signalling pathways. methods: the ethics committee (ceua -comissão de ética no uso de animais -ufrgs; nr. 29,818) approved all animal procedures and experimental conditions. male wistar rats of 3-and 21-month-old were used, and plasma was obtained from the trunk blood. evs were isolated with exoquick following the manufacturer's instructions. microrna was isolated from evs and then amplified. microrna was labelled using the flashtag biotin hsr rna labelling kit and profiled on affymetrix genechip microrna 4.0 arrays. ingenuity pathway analysis (ipa) was used to identify pathways regulated by significantly altered micrornas. results: microarray analysis revealed 728 micrornas. of these micrornas, 48 were differentially expressed between aged and young-adult animals, 18 micrornas were significantly upregulated and 30 were downregulated in aged animals compared to young adult (p < 0.05; fold change of |1.1|). a conservative filter was applied on ipa and only experimentally validated and highly conserved predicted mrna targets for each microrna was used. ipa analysis showed that cardiac hypertrophic signalling is ranked as highly predicted targets for these differentially expressed micrornas (p < 0.0001). moreover, ipa demonstrated that this canonical pathway is upregulated in aged animals when compared to young adult. in addition to cardiac hypertrophic signalling, other relevant cardiovascular canonical pathways, such as endothelin-1 signalling and intrinsic prothrombin activation pathway have predicted targets. summary/conclusion: our results showed for the first time that micrornas profile in circulating evs has a potential role to drive heart senescence and consequent cardiac diseases which represents the leading cause of death. introduction: introduction: the vascular endothelium and smooth muscle form adjacent cellular layers that comprise part of the vascular wall. here, we examined the extent to which extracellular vesicles (evs) vesicles participate in endothelial-vascular smooth muscle cell communication. methods: methods: evs were collected from rat aortic endothelial and smooth muscle cell serumfree media by ultracentrifugation. vesicle morphology, size and concentration were evaluated by transmission electron microscopy and nanoparticle tracking analysis. endothelial cell and vascular smooth muscle cell cultures were subjected to various concentrations of evs for various times. functional assays were performed. results: results: western blot as well as shot gun proteomic analyses revealed sets of proteins common to both endothelial-and smooth muscle-derived ev as well as proteins unique to each vascular cell type. functionally, endothelial-derived evs stimulated vascular cell adhesion molecule-1 (vcam-1) expression and enhanced leukocyte adhesion in vascular smooth muscle cells while smooth muscle evs did not elicit similar effects in endothelial cells. evs from endothelial cells also induced protein synthesis and senescenceassociated β galactosidase activity in vascular smooth muscle cells. proteomic analysis of vascular smooth muscle cells following exposure to endothelial cellderived evs revealed upregulation of several proteins including pro-inflammatory molecules, high-mobility group box (hmgb) 1 and hmgb2. pharmacological blockade of hmgb1 and hmgb2 and sirna depletion of hmgb1 in smooth muscle cells attenuated nfkb (p65) phosphorylation and nuclear translocation, vcam-1 expression and leukocyte adhesion induced by endothelial cell evs. summary/conclusion: conclusions: these data suggest that endothelial cell-derived evs can enhance signalling pathways that induce a pro inflammatory in vascular smooth muscle cells. introduction: graft patency is one of the major determinants of long-term outcome following coronary artery bypass graft surgery (cabg). biomarkers, if indicative of the underlying pathophysiological mechanisms, would suggest strategies to limit graft failure. many studies have generated compelling data on the sensitivity of mvs as biomarkers of cardiovascular disease progression and events. the mv usefulness in cabg has been tested only in a study that highlighted their importance in surgical haemostasis. no information is so far available on the association between the amount or pattern of circulating mvs and cabg outcome. we aimed to evaluate whether mv pre-operative signature could predict mid-term graft failure. methods: this was a nested case-control substudy of the coronary bypass grafting: factors related to late events and graft patency (cage) study that enrolled 330 patients undergoing elective cabg. of these, 179 underwent coronary computed tomography angiography 18 months post-surgery showing 24% graft occlusion. flow cytometry mv analysis was performed in 60 patients (30/group with occluded [cases] and patent [controls] grafts) on plasma samples collected the day before surgery and at follow-up. results: before surgery, cases had two-fold (p = 0.020) and four-fold (p = 0.042) more activated platelet-derived and tf+ mvs, respectively than controls. the mv thrombin generation capacity was also significantly greater (p < 0.05). this mv signature predicted graft occlusion (auc of 0.897 [95%ci: 0,77-0,96], p = 0.02). by using a mv-score (0-6), the or for re-occlusion for a score above 3 was 16.3 (95% ci 4.1-65.3, p < 0.001). summary/conclusion: the pre-operative signature of mvs is an independent predictor of mid-term graft occlusion in cabg patients and a cumulative mvscore stratifies patient's risk. since the mv signature mirrors platelet activation, patients with a high mvscore would benefit from a personalized antiplatelet therapy. exosomes from engineered immortalized human heart cells improve ventricular function and attenuate fibrosis in mice with arrhythmogenic cardiomyopathy yen-nien lin, lizbeth sanchez, rui zhang, thassio ricardo ribeiro mesquita, chang li, ahmed ibrahim, eduardo marbán and eugenio cingolani heart institude, cedars sinai medical center, los angeles, usa introduction: arrhythmogenic cardiomyopathy (ac) is characterized by progressive loss of cardiomyocytes and fibrofatty tissue replacement. currently, there is no effective treatment for this disease. exosomes (imexos) secreted by heart stromal cells, engineered to be immortal and overexpressing β-catenin, exert anti-inflammatory and anti-fibrotic effects and improve ventricular function in models of ischaemic injury (ibrahim et al., nature bme 2019). methods: to investigate the effectiveness of imexos in a murine model of ac, four-week old homozygous dsg2 knockout (dsgko) mice and wild type (wt, age-and strain-matched) mice were compared. dsgko mice were randomized to receive weekly imexos or vehicle via intravenous injection for 4 weeks. neonatal rat ventricular myocyte (nrvm) proliferation and apoptotic assays were performed to explore potential effects of exosomes. results: biodistribution studies of dir-labelled imexos revealed some cardiac uptake, along with strong signals in spleen. at 4 weeks, dsgko mice which had received intravenous imexos showed improved cardiac function (echocardiographic ejection fraction 73 ± 2 vs 59 ± 5% in vehicle mice, p = 0.03), with an underlying attenuation in myocardial fibrosis by histology. electrophysiology test showed shorter qrs duration (4.0 ± 2.0 ms imexo vs 6.5 ± 2.9 ms vehicle, p = 0.02) and effective refractory period. programmed ventricular stimulation showed dsgko mice which had received imexos were remarkably less prone to ventricular tachycardia induction (10.0 ± 32% vs 55.0 ± 52% in vehicle, p = 0.031). in vitro study showed nrvm exposed to imexos for 2 days exhibited higher brdu expression relative to vehicle group, and less annexin-v expression after oxidative stress induced by 10-minute illumination with 254 nm uv. summary/conclusion: intravenous administration of imexos improved cardiac function, reduced cardiac fibrosis, and suppressed arrhythmogenesis in ac. our findings motivate clinical testing of imexos in ac, an orphan disease with great unmet medical need. funding: nih r01 hl124074 (to em) cardiac-derived extracellular vesicles contribute to communication between heart and brain in chronic heart failure (chf) and target nrf2/ are signalling changhai tian a , lie gao b and irving zucker b a department of cellular and integrative physiology, university of nebraska medical center, omaha, usa; b department of cellular and integrative physiology, university of nebraska medical center, omaha, usa introduction: mirnas regulate the translation of proteins that are involved in redox homoeostasis in the heart and brain. intra-and/or inter-organ communication takes place by multiple mechanisms including extracellular vesicular (ev) transport. our previous studies suggested that cardiac derived mirna-enriched evs contribute to the dysregulation of nrf2/antioxidant enzyme (are) signalling in the myocardium via intercellular cross-talk, and result in the decreased nrf2/are signalling in the sympatho-regulatory areas of the brain in chf. however, it is unclear if cardiac derived evs circulate to the central nervous system evoking sympatho-excitation by disrupting central redox homoeostasis. methods: cardiac-specific membrane gfp+ mice were generated to track the brain distribution of cardiac evs in rats with chf (coronary ligation). the isolation and characterization of evs were carried out by differential ultracentrifugation, tem, nanosight, western blotting, and qrt-pcr. transfection, labelling, and microinjection of evs into the rostral ventrolateral medulla (rvlm) were performed. results: nrf2 protein was reduced in the rvlm of chf rats consistent with an upregulation of nrf2-targeting mirnas. nrf2-targeting mirnas were enriched in cardiac and circulating evs of chf rats. nrf2-targeting and cardiac-specific mirnas were abundant in brain-derived evs. circulating evs were taken up by neurons in sympatho-regulatory areas of the brain. mirna-enriched evs from chf animals increased sympathetic tone which was prevented by a cocktail of nrf2-targeting mirna inhibitors. summary/conclusion: myocardial infarction-induced mirna-enriched evs mediate the inter-organ crosstalk between heart and brain in the oxidative regulation of sympathetic outflow through targeting the nrf2/ are signalling pathway. these findings suggest that cardiac-derived ev mirnas targeting nrf2/are signalling may act as an endocrine signalling mediator of chf that has potential as a novel therapeutic target. introduction: a fine-tuned communication between cardiac cells is vital to maintain myocardial integrity and contractility. not only an impairment of gap junction (gj)-mediated intercellular communication, but also defects in ev-mediated communication have been associated with ischaemic heart disease, a major causative factor of heart failure. we have previously shown that cx43, the main ventricular gj protein, assembles into channels at the evs surface, mediating the release of vesicle content into target cells.the main objective of this work was to characterize the signals underlying protein sorting into extracellular vesicles (evs) in a human pathophysiological context, using connexin43 (cx43) as a model substrate. methods: animal models of ischaemia/reperfusion (i/ r) injury by ligation of the left anterior descending coronary artery, ex vivo and in vitro ischaemia models and human patients were used to investigate the secretion of ev-cx43. results: release of cx43 was downregulated in circulating vesicles from i/r-injured mice and patients with st-segment elevation myocardial infarction, as well as in intracardiac and cardiomyocyte-derived evs. additionally, we show that ubiquitin signalled the release of cx43 in basal conditions but appeared to be dispensable during ischaemia. depletion of the autophagy adaptor p62 partially restored the secretion of cx43, suggesting an interplay between ischaemiainduced cx43 degradation and secretion. summary/conclusion: overall, we demonstrated that ischaemia impairs the sorting of cx43 into evs, which may ultimately affect long-distance communication. through the identification of the underlying molecular mechanisms and players, these results pave the way towards the development of innovative diagnostic and therapeutic strategies for cardiovascular disorders. introduction: remote ischaemic conditioning is a cardioprotective intervention which protects the heart against ischaemia/reperfusion injury. transient activation of toll-like receptor 4 (tlr4) and its downstream regulators (tnfα and il-6) have been implicated in cardioprotective interventions. extracellular vesicles (evs) play a role in cardioprotection through the activation of the tlrs. however, since isolation of evs in high amounts with suitable purity from blood is a challenge, our aim was to develop a cellular model system from which tlr-inducing, cardioprotective evs can be isolated in a reproducible manner. methods: ev release from hek293 cells was induced by calcium-ionophore a23187. evs were characterized, cytoprotection by evs against simulated ischaemia/ reperfusion injury and its mechanism were investigated in h9c2 and ac16 cell lines. results: a23187 induction of hek293 cell induced ev release and the isolates contained mostly large evs. evs decreased cytotoxicity and apoptosis due to 16 h ischaemia followed by 2 h reperfusion in h9c2 and ac16 cells in a dose-dependent manner. evs activated tlr4 and its downstream signalling pathway in h9c2 and ac16 cells as well as the expression of cytoprotective haem oxigenase 1 (ho-1) in h9c2 cells. summary/conclusion: a23187-induced evs exert cytoprotection in h9c2 and ac16 cells by inducing tlr4 signalling and ho1 expression. therefore, evs released via calcium-ionophore treatment may serve as a basis of an efficient carpdioprotective therapy. introduction: biliary strictures may be benign or malignant. the major malignant causes of biliary stricture are a primary cholangiocarcinoma (cca) or pancreatic ductal adenocarcinoma (pdac). there is ongoing debate about adequate diagnostics in biliary strictures of unknown aetiology. micrornas (mirnas) are small non-coding rnas important in tumourigenesis. mirna have been found to be enriched in exosomes, small membrane-bound extracellular vesicles (ev) of endocytic origin, which is a novel pathway for intercellular signalling within the tumour microenvironment and have been implicated in loco-regional pre-metastatic niche formation. this project aims to investigate circulating-free and ev mirnas as biomarkers that can aid diagnosis in patients with a biliary stricture. we will (1) isolate and characterise evs in plasma and bile from patients with benign and malignant biliary strictures (i.e. pancreaticobiliary cancers); and (2) identify differentially expressed circulating-free and ev mirnas in plasma and bile suitable for detecting malignancy. methods: sample size (n = 126) was calculated for a study power of 90% and α error of 5% for the ability of extracellular mirnas to discriminate benign from malignant biliary strictures. prospective matched plasma and bile samples will be collected from patients with benign (n = 63) and malignant (n = 63) biliary strictures undergoing endoscopic retrograde cholangiopancreatography (ercp). evs will be isolated from the biofluids by ultracentrifugation and/or size exclusion chromatography and then characterised (tem, nta and immunoblotting). circulating-free and ev-associated mirnas will be profiled using small rna sequencing. extracellular mirna "signatures" will then be validated by rt-qpcr, and diagnostic accuracy confirmed (sensitivity, specificity, auc). results: evs derived from patient samples have been characterised using nta, western blotting and tem. sec derived evs appear to be more well-defined than uc evs with marker positivity for cd63, cd81 and cd9. ongoing work will be focused on rna profiles of evs from both malignant and benign cohorts. summary/conclusion: there is currently no effective method to differentiate benign from malignant biliary strictures. novel plasma and bile circulating-free and ev-associated mirna biomarkers may improve the speed and accuracy of diagnosis, resulting in considerable patient benefits. furthermore, as little is known about the ev-associated function of these tumours, candidate ev-mirnas could be taken from "bedside to bench" and their function further investigated using in vivo, vitro and silico models. introduction: urine is a source of extracellular rna (exrna) biomarkers that can be obtained non-invasively throughout pregnancy. several studies have profiled extracellular mirnas in biofluids during pregnancy, but few have profiled extracellular mrnas (ex-mrnas) in urine. objective: to optimize methods for ex-mrna isolation and rna-seq library preparation from urine of healthy pregnant and non-pregnant females. methods: rna was isolated from pooled non-pregnant urine using kits based on ev precipitation (mircury exosome kit for csf/urine, seramir), ev affinity purification (exorneasy), and protein precipitation (mirneasy serum/plasma advanced). next, long (>200nt) and short rnas were isolated from ev enriched urine of pregnant (n = 5) and non-pregnant (n = 5) individuals using the mircury kit followed by the mirneasy micro kit. rna-seq libraries were prepared using the smart-seq v4 ultra low input rna (oligo(dt) priming) and the smarter stranded total rna-seq kit v2 -pico input (random priming) methods (takara). preliminary data were obtained using the illumina miseq, and aligned using star v.2.7.3.a. results: overall, rna isolation using mircury followed by the smart-seq v4 library preparation kit yielded the highest % of mapped reads: 42% in pooled non-pregnant, 27% in individual non-pregnant, and 31% in individual pregnant urine. for rna extracted using the mircury kit, the smart-seq v4 libraries had higher % of mapped mrna reads compared to pico libraries (p < 0.05, t-test). in contrast for mirneasy advanced it was reversed (38% vs 21%). summary/conclusion: early results from low-depth sequencing show the highest mrna mapping rates for mircury followed by the smart-seq v4 kit. high-depth sequencing data are now being generated, which will enable us to perform detailed comparisons of different rna species from the rna profiles obtained using different library preparations and rna isolation methods from urine of pregnant and non-pregnant subjects. funding: this study was funded by nih 7 k99 hd096125-02, nih u01 hl126494, and a ucsd igm-illumina mini-grant. il-2 mutein-induced changes of exosomal mirna cargo in a humanized mouse model emily lurier, erik sampson, patrick halvey, mike cianci and katalin kis-toth pandion therapeutics, cambridge, usa introduction: regulatory t cells (tregs) are key contributors to immune homoeostasis. decreased number and/or function of these cells are frequent features of many autoimmune diseases linked to the development of tissue inflammation. while interleukin-2 (il-2) is essential for pan t cell proliferation and performance, low dose il-2 treatment has been shown to preferentially affect tregs and is being evaluated as an intervention in autoimmune diseases. pt101 is a novel il-2 mutein fc fusion molecule (il-2 m) designed to selectively engage with tregs. using a humanized nod-scid il2rn-null (nsg) mouse model we have shown that pt101 expanded tregs without significant effects on other immune cells. we have also shown that tregs from pt101-dosed humanized mice exhibit increased expression of foxp3 and cd25, and demethylation of foxp3 and ctla-4 genes, suggesting enhanced function and stability. in the current study we investigated the mirna content of plasma exosomes isolated from pt101-or vehicle-treated mice in order to identify treg specific mirnas from the il-2 m treated animals. methods: cd34+ haematopoietic stem cell humanized nsg mice were dosed once subcutaneously with pt101 or vehicle. plasma samples from 8 mice were collected at day 7 and exosome isolation was conducted using the exoquick method. small rna was extracted and quantified using the bioanalyzer small rna assay. an illumina nextseq instrument was used for library preparation and sequencing with 75bp single end reads at an approximate depth of 10-15 million reads per sample. raw sequences were mapped to human genome grch37 and analysed via a pipeline provided by the university of california santa cruz. results: rna within the exosomes from vehicle and il-2 m-treated groups was mostly comprised of mirna and trna. plasma was pooled from 8 animals per treatment group and differential expression was determined using a twofold change cut-off. we found that pt101 treatment actively altered the mirna content of plasma exosomes, compared to exosomes from vehicle-treated mice. many of the differentially expressed mirnas are involved in immunoregulation. summary/conclusion: plasma exosomes from pt101treated humanized mice encapsulated treatment-specific mirnas which can potentially be used as systemic biomarkers of treg expansion and function. identification of potential biomarkers in microglial specific exosomes isolated from prion-infected serum introduction: transmissible spongiform encephalopathies (tse) are neurodegenerative disorders caused by the misfolding of the cellular prion protein (prpc) to the beta-sheet rich abnormal prion protein (prpsc). prpsc aggregates in the brain and causes amyloid plaques, neuronal loss, spongiform degeneration and microglial activation. currently, definitive diagnosis of tse diseases is only confirmed post-mortem thus a diagnostic test in accessible body fluid is of interest. exosomes are a good resource for biomarker discovery since they cross the blood-brain barrier easily and contain protein, lipids and nucleic acids from the cells of origin. the goal of this study was to look at biomarkers from brain-originating exosomes (specifically microglia) isolated in the serum of prion-infected animals. methods: westerns and nanoparticle tracking analysis (nta) were used to look at the composition of microglial-specific exosomes. as proof of principle, exosomes were isolated from a microglial cell line (bv2 cells). a cd63 antibody was labelled with a fluorophore and binding to exosomes was visualized via nta. exosomes were isolated from serum of both prioninfected and mock-infected mice throughout disease course. a macrophage specific antibody (f4/80) was bound to beads which were used to isolate exosomes which includes those of microglial origin. microrna was extracted from these exosomes and next-generation sequencing (ngs) was performed using the illumina platform. clc genomics workbench was used for bioinformatics analysis. results: microglial and macrophage proteins (tmem119 and iba1) were identified in exosomes isolated from bv2 cells and prion-infected mouse serum. macrophage exosomes were isolated via a novel antibody-bead based system. results of the ngs analysis of the microrna isolated from these exosomes indicated a series of mirna that could differentiate between control and infected samples as well as age-specific markers. summary/conclusion: to our knowledge, this is the first time microglial-specific exosomes have been isolated from prion-infected serum from early and end stage disease. the results of this analysis could facilitate the diagnosis of prion disease in easily-accessible biofluids pre-mortem. comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease introduction: urinary extracellular vesicles (uevs) are emerging as a source for early biomarkers of kidney damage, holding the potential to replace the conventional invasive techniques including kidney biopsy. several methods are available for uev isolation. our aim was to compare different workflows and isolation by hydrostatic filtration dialysis (hfd), ultracentrifugation (uc) and a kit based isolation method for their subsequent use in mirna-seq and rna-seq for biomarker discovery in diabetic kidney disease. methods: type 1 diabetic patients (t1d) with macroalbuminuria and normoalbuminuric healthy controls were included in the study. sample collection and all experiments were performed in accordance with the declaration of helsinki. evs were isolated from 10-50 ml of 24 h urine collections by uc, hfd, or a commercially available kit (purification based on spin column chromatography, urine exosome purification and rna isolation midi kit, norgen biotech, canada) each with different established urine clarification steps. quality control of the evs was performed with negative staining em, nta and western blotting. isolated rnas were profiled with bioanalyzer pico kit and subjected to mirna and mrna sequencing. for rna-seq, cdna library was prepared using smart-seq v4 ultra low input rna kit for sequencing (takara bio, japan). rna-seq was performed using hiseq 2000 (illumina). mirna-seq library was prepared using qiaseq mirna library kit (qiagen, germany). mirna-seq was performed on the illumina hiseq 4000 platform (illumina). results: our data showed that uev yield, morphology and size distribution were closely similar in hfd and uc preparations, while lower yields were obtained using the kit. by western blot, ev markers were detectable in samples isolated by hfd and uc but not readily in samples isolated with the kit. tamm-horsfall protein was detected in all the samples and albumin levels appeared higher in hfd and kit isolated samples relative to uc samples. the number of paired-end reads for rna-seq in hfd and uc samples (in both > 5 m) were closely similar. instead, rna reads were lower than 2 m for the kit samples. for mirna-seq, the number of reads as well as the molecular biotype distribution were similar for the three methods. by principal component analysis of the rna-seq data, we observed that hfd and uc grouped together showing similarities. however, for mirna-seq data such similarities were not obvious. this suggests that the three different workflows and isolation principles may enrich different mirna-rich uev preparation components. summary/conclusion: our transcriptomics data shows that hfd and uc are suitable methods to isolate uevs for mirna-seq and rna-seq. the kit based method appears better suited for mirna-seq. introduction: exosomes contain a variety of biomolecules including dna. knowledge of cfdna distribution and localization in bioliquid is important for understanding both biological function of cfdna and exosomes. some publications state that a large proportion of plasma cfdna is localized in exosomes. to quantify cfdna content in free vs. exosomal form in human plasma, urine, and saliva, we employed subx technology, which allows affinity capture dna via phosphates groups of the polynucleotide chain and exosomes via membrane surface phosphate moiety clusters. subx is a proprietary compound that can simultaneously bind to both cfdna and exosomes in bioliquids, thus allowing precipitation of the [subx-dna/ subx-exosomes] complexes without ultracentrifugation. methods: detection of subx-dna and exosomes binding was done by measurement of particle sizes using zetasizer nano zs and nanosight ns300. the samples were processed with the subx exo-dna isolation kit following the standard protocols. dna, protein and lipid concentrations were measured by fluorescent assays using qubit fluorometer. results: subx efficiently and selectively captures and co-precipitates cfdna and exosomes directly from bioliquids. exosomes are easily extracted from the pellet in exosome reconstitution buffer (erb), followed by subsequent isolation of tightly bound cfdna from the subx pellet. erb does not extract dna form the [subx -dna] pellet and thus does not contaminate reconstituted exosomes with cfdna. thus, we separate two distinct types of extracellular materialintact exosomes and purified cfdna in a single protocol from the same sample. over 90% of dna in plasma and urine exist as a free circulating pool, while in saliva up to 30% is associated with exosomes. thus, cfdna distribution is probably bioliquid-specific and must be evaluated by methods that eliminate cfdna-outer exosomal membrane aggregation. summary/conclusion: subx technology is suitable for simultaneous isolation of both cfdna and exosomes from the same bioliquid sample. subx separates cfdna fragments non-specifically attached to the outer lipid layers of the exosome membrane from the true intra-exosomal cfdna. in contrast, salting-out peg technique is associated with aggregation of macromolecules and vesicles and thus leads to overestimation of exosome-associated polymers content, including cfdna. tracing extrachromosomal dna inheritance patterns in glioblastoma using crispr eunhee yi, amit gujar, hoon kim, albert cheng and roel verhaak jackson laboratory for genomic medicine, farmington, usa introduction: glioblastoma multiforme (gbm) is the most lethal brain tumour; it is characterized by poor response to standard post-resection radiation and cytotoxic therapy, resulting in a dismal prognosis with a five-year survival rate of 10%. recurrence after therapy for gbm is unavoidable. there are substantial differences among the cells of gbm tumours in the abundance and types of genetic material. this heterogeneity likely is the major cause of therapy failure, the development of treatment resistance, and ultimately recurrence. a recent study has suggested that the amount of a particular type of dnaextrachromosomal dna (ecdna)differs substantially among different gbm tumours, and differs within a given gbm tumour over time. despite the speculation that ecdna is a key factor of tumour heterogeneity, how ecdna is propagated and distributed amongand how it behaves withincancer cells is completely unknown. methods: to address this gap in knowledge, this study focused on developing a novel cytogenetic crisprbased tool that enables visualization and tracking ecdna behaviour in live gbm cells. results: we found breakpoint sequences resulting from genome rearrangements during ecdna formation by performing computational analysis from whole genome sequencing data. and each breakpoint was regarded as a unique target sequence for ecdnaspecific labelling. the uniqueness of each breakpoint was validated by breakpoint-pcr (bp-pcr). furthermore, the location and the amount of each breakpoint were observed by breakpoint-fish (bp-fish) analysis in gbm cells. summary/conclusion: this results will be strong evidence to make ecdna-specific crispr system in further research. tracing ecdna dynamics will provide new insight into the impact of ecdna on cancer evolution. introduction: small extracellular vesicles (sevs) are 30-150 nm vesicles that mediate intercellular communication by transferring rna and proteins to the recipient cells. these cargo molecules are selectively sorted into sevs and mirror the physiological state of the donor cells. given that sevs can cross the bloodbrain barrier and their composition can change in neurological disorders, there is an increasing interest in elucidating the molecular signatures of sevs in circulation as disease biomarkers. however, circulating sevs are derived from multiple cellular sources and determining their source is challenging. information on sev composition can be beneficial in predicting whether these sevs are released predominantly from central nervous system cells. we hypothesized that differentially expressed mirnas between neuronal sevs and astrocytic sevs could be used as cell-typespecific signatures. methods: small extracellular vesicles were isolated from cell culture media of postnatal mouse primary neurons and astrocytes using differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna from neurons, astrocytes, and their respective sevs were used for transcriptome and small rna sequencing. results: we observed that only a subset of cellular mirnas was packaged into sevs; differential expression of specific mirnas between sevs and their corresponding cells suggest that cells employ special mechanisms to sort mirnas into sevs. these mechanisms could be celltype specific since neuronal sevs showed a different mirna profile compared to astrocytic sevs. exomotifs, the short sequence motifs that control the loading of rna into sevs, were present in differentially expressed mirnas. we also observed that five rnabinding proteins, which are associated with passive or active rna sorting into sevs, were differentially expressed between neuronal and astrocytic cells. summary/conclusion: mirna signatures of sevs from neurons and astrocytes could be beneficial in determining if these cell types contribute to the alterations of sev composition in circulation in neurological disorders. cell-type-specific selectivity in rna loading might be attributed to the differential expression of rna-binding proteins. introduction: analytes present in the extracellular fraction of bodily fluids (ex. blood, urine) have utility as a tool for uncovering the molecular landscape of tumours and hold great potential for discovery of individualized cancer medicine. urine, being noninvasive as a sample type, has an obvious advantage over blood when used for liquid biopsy purposes. however, potential for microbial proliferation and the labile nature of host cells and extracellular vesicles (evs) at the point of sample collection/transport to the lab drives the need for stabilization of urine samples. development of such sample stabilization opens up capability for the detection of various biomarkers present in the extracellular fraction to be used in liquid biopsy. this is of particular concern as studies around urinary analytes for cancer diagnosis, progression and therapeutic effect are rapidly expanding in cohort sizes. multi-site collections and at-clinic collections are increasingly prohibitive for large scale recruitment and also lead to variability in the time between collection and processing. methods: in this study, we have analysed two commercially available ev extraction kits and compared them with ultracentrifugation technique for size, concentration and specificity of the isolated evs from human urine samples with and without our proprietary preservation solution using nanoparticle tracking analysis and western blot analysis for exosomal membrane markers. ev rna contents in various urine fractions (first morning first void, random first void and midstream) were compared using rt-qpcr assay to provide better understanding of the collection techniques and fractionations that are ideal for ev research work. results: in our current work, we have bench-marked human urine collection and ev extraction in order to provide recommendations in standardization of sample acquisition and processing for urinary ev studies. we have utilized these standardization in order to develop a novel and efficient sample stabilization principle for preservation of evs and ev rna in urine samples during an ambient temperature hold. summary/conclusion: taken together, we have established a framework for evaluating technologies and techniques in the ev sample processing space, which can be utilized by other research groups. vn96-isolated plasma extracellular vesicles improve tumour mutation detection by next-generation sequencing compared to cell-free dna and correlate with tissue biopsy of nsclc patients introduction: liquid biopsy is a minimally-invasive diagnostic method that detects circulating biomarkers and has the potential to improve access to molecular profiling for nsclc patients when tissue biopsy material is unavailable or insufficient. although isolation of cell-free dna (cfdna) from plasma is the standard liquid biopsy method for detecting dna mutations in cancer patients, the sensitivity can be highly variable. vn96 is a 27 amino acid peptide with an affinity for heat shock proteins that are exposed on the surface of extracellular vesicles (evs); peptide-ev aggregates readily sediment using a benchtop centrifuge and therefore the vn96 peptide provides a rapid, clinically-amenable procedure for ev isolation. in this study, we determine whether isolation of evs from nsclc patient plasma improves the sensitivity of single nucleotide variants (snvs) detection compared to cfdna and correlate genetic changes observed by liquid biopsy with tumour ffpe tissue biopsy. methods: blood was collected from stage iii/iv nsclc patients with informed consent in either edta or cell-free dna bct® collection tubes and plasma was harvested within 30 minutes. total nucleic acid (tna) was extracted from either vn96-isolated evs from edta plasma or directly from plasma collected in edta or cell-free dna bct® tubes (cfdna). snvs were detected by next-generation sequencing (ngs) results: vn96 isolation of evs from plasma resulted in higher recovery of dna than cfdna isolation. the snvs detected in both ev-dna and cfdna correlated well with those reported in matched ffpe tumour tissue using ngs, including 100% specificity for egfr mutations. no improvement in snv detection was observed using cell-free dna bct® collection tubes compared to edta tubes. isolation of evs with the vn96 peptide prior to sequencing improved a number of ngs parameters including library yield, total reads, median read coverage and molecular coverage, resulting in improved sensitivity of snv detection. summary/conclusion: in summary, our research demonstrates that vn96-based ev isolation is useful for molecular profiling of nsclc patients for whom tissue biopsy is not an option, thereby improving access to molecular profiling and targeted therapies. funding: atlantic canada opportunities agency novel markers for neuroendocrine prostate cancer divya bhagirath a , michael liston b , theresa akoto a and sharanjot saini a a augusta university, augusta, usa; b veteran affairs, san francisco, usa introduction: prostate cancer (pca) is fuelled by androgens and androgen receptor (ar) signalling. therefore, ablation of ar signalling by androgen deprivation therapy (adt) is the goal of first-line therapy that results in cancer regression initially. however, two to three years post-adt, the disease develops into castration-resistant prostate cancer (crpc). as a second-line of therapy, next generation of ar pathway inhibitors (api) such as enzalutamide (enz) are used that are effective initially followed by emergence of drug resistance. a subset of api-resistant tumours emerges to an ar independent state via undergoing a trans-differentiation to neuroendocrine lineage, a process referred to as neuroendocrine differentiation (ned). due to lack of ar signalling, these pca variants, referred to as neuroendocrine prostate cancer (nepc), are impervious to anti-androgen therapy and constitute an aggressive variant of advanced crpc with poor prognosis. currently, there is a lack of effective molecular biomarkers for predicting api therapy resistance and emergence of therapy-induced ned. methods: exosomes/evs were isolated from sera of a patient cohort with/without ned. the study was conducted in accordance with ethical guidelines of us common rule and was approved by the institutional committee on human research. written informed consent was obtained from all patients. following extensive characterization of evs by electron microscopy, nanosight tracking analyses and western blotting of exosomal markers, small rna sequencing was carried out on illumina hiseq platform to identify differentially expressed transcripts. machine learning algorithms were applied to clinical sequencing data to train a "mirna classifier". further, we probed the proteomic profile of exosomes isolated from nepc cellular model nci-h660 and enzalutamide resistant crpc cell lines by mass spectrometry. results: we identified that transition from crpc-adenocarcinomas to neuroendocrine states is associated with significant ev-mirna dysregulation, with a specific dysregulation in certain mirna families. with the application of machine learning algorithm, we identified an ev-based "molecular classifier" that can robustly stratify crpc-ne tumours from crpc-adenocarcinomas. proteomic analyses identified novel nepc-specific, glycosylated proteins that can be exploited for nepc diagnosis. summary/conclusion: our data suggest that ev mirna and protein profile can predict neuroendocrine differentiation in advanced castration-resistant prostate cancer patients. exosomal mrna in diagnosis strategy for hepatocellular carcinoma aleksandr abramov, alisa petkevich, vadim pospelov and pavel ogurtsov peoples' friendship university of russia (rudn university), moscow, russia introduction: exosomal cargo is informative source illustrating the genetic events happening in cells, what can be especially advantageous in case of cancer development for disease progression or treatment effectiveness monitoring. methods: 10 plasma samples of hepatocellular carcinoma (hcc) patients, 10 plasma samples of patients with liver cirrhosis 3-4 on the hepatitis c virus (hcv) background, 5 healthy donors' plasma samples. exosomes were isolated with ultracentrifugation, western blot (cd63, cd9) was performed. total mrna was isolated with exosomal rna isolation kit, norgen biotec corp. sequencing was carried out on a minion sequencer. housekeeping genes (gapdh, b2 m, actb, tuba1a). detected mutations were confirmed by real-time pcr with specific highly sensitive lna probes. results: significant changes in expression levels were identified for 50 genes in hcc and liver cirrhosis groups (increasing up to x200 compared to control samples and decreasing up to no detected expression). in 6 out of 10 patients with hcc mutant burden was significant increased compared to mutant burden in groups with cirrhotic samples. in 8 out of 10 patients with hcc increased expression for mrna line-1 was identified compared to cirrhotic patients. summary/conclusion: exosomal mrna expression levels may serve as a prognostic and diagnostic marker for patients with liver cirrhosis caused by hcv for hcc risk development. funding: research is supported with federal funds "5-100" circulating extracellular vesicle signatures in small cell lung cancer michela saviana a , giulia romano a , giovanni nigita b , robin toft a , patricia le a , kai wang c , mario acunzo a and patrick nana-sinkam a a virginia commonwealth university, richmond, usa; b the ohio state university, columbus, usa; c institute for systems biology, seattle, usa introduction: lung cancer is the leading cause of cancer deaths worldwide and classified primarily as either non-small cell lung cancer (nsclc) or small cell lung cancer (sclc). compared to nsclc, sclc has a faster growth rate, earlier widespread metastasis, and shorter overall survival. the early diagnosis of sclc and the development of novel therapeutics have proven challenging. thus, progression and recurrence rates remain high. non-invasive methods for cancer detection are increasingly being used to inform clinical decision making. extracellular vesicles (evs) have recently emerged as potential carriers of genetic contents such as micrornas (mirs) to induce reprogramming of components of the microenvironment in cancer initiation and progression. moreover, extracellular mirs expression profiles have been shown to have signatures related to tumour classification, diagnosis, and progression. methods: we selected a cohort of patients divided into 4 groups: high-risk smokers, adenocarcinomas, squamous carcinomas, and sclc. we extracted total circulating ev and plasma rna from plasma (38 patients in total) and rna from plasma in a separate group (24 patients in total). utilizing both next-generation sequencing (ngs) and nanostring platforms, we analysed for global microrna (mirs) expression patterns. candidate mirs were then validated by qrt-pcr. results: we identified several deregulated mirs in both evs and plasma of sclc patients compared to the other groups. for evs, we validated mir-1285-5p as a significant biomarker for the late stage of sclc compared to controls. in the case of plasma, we validated the upregulation of mir-375 in sclc compared to controls. summary/conclusion: our results indicate that a potential combination of plasma (mir-375) and ev-based (mir-1285-5p) mirs be valuable biomarkers for sclc detection and serve as a basis for a non-invasive sclc classifier. funding: virginia commonwealth university, doim -nih/nci introduction: the isolation of evs from milk is technically challenging due to the complexity of milk. currently used separation procedures allow for the removal of milk fat globules and cells (by low speed centrifugation of fresh milk), removal (by acidification), or disruption (by addition of edta) of casein micelles. using these protocols the integrity, composition and targeting of bovine milk evs has been evaluated and has led to believe that milk evs might withstand these conditions. however, the effects on functionality of milk evs (i.e. immunomodulatory properties) after processing and isolation have not been studied. therefore, we have set up an in vitro culture system using a human t cell line that allows for the rapid screening of milk ev functionality. methods: fresh bovine milk was defatted and cells were removed after 2x3,000 g centrifugation, followed by differential centrifugation at 5,000 g and 10,000 g. this milk was either subjected to acidification with hcl, or edta was added, or the milk supernatant remained untouched. top down optiprep density gradient separation followed by sec was used to further purify evs. these highly purified milk evs were added to human jurkat t cells, which were simultaneously stimulated using anti-cd3 and anti-cd28 antibodies. after 24 h t cell activation was measured by il-2 cytokine production. results: precipitation or disruption of casein micelles allowed for the substantial removal of proteins during isolation compared to directly isolated evs, which aids in the purification of milk evs. in vitro analysis revealed that in the presence of directly isolated, or edta isolated milk evs, jurkat cells were suppressed in their activation as measured by il-2 production. remarkably, evs isolated from hcl-acidified milk were impaired in their suppressive capacity to inhibit il-2 production. summary/conclusion: although casein removal from bovine milk greatly improves purity of isolated milk evs, the detrimental effects on ev functionality should be considered. interestingly, evs exposed to acidic conditions lost their ability to modulate t cell activation, which is in contrast with the general believe that milk evs could withstand the gastro-intestinal tract. funding: this work is funded by the european union's horizon 2020 framework programme under the grant fetopen-801367 evfoundry. optimising methods for separation and characterisation of extracellular vesicles from skim milk and infant milk formula introduction: infant milk formula (imf) is intended to impart nutrition to infants, similar to breast milk. however, although industrial imf production involves harsh treatment, potential consequences on extracellular vesicles (ev) in imf are not yet established. this study aimed to optimise methods for separating evs from imf and skim milk (sm) and to characterise the evs in accordance with misev2018. methods: sm and imf were either not treated (nt) or treated with acetic (aa) or hcl acid (isoelectric precipitation, ip), to remove caseins. samples were then subjected to differential ultracentrifugation (duc) or gradient ultracentrifugation using iodixanol solution (guc). for duc, 38 ml samples were centrifuged at 12 k g, 35 k g, 75 k g, 100 k g and 200k g sequentially for 75 min each and pellets re-suspended in 1 ml pbs. preparation of agarose microspheres for high-efficient separation of extracellular vesicles cheng-tai chen, chien-an chen, carolyn yen and nien-tzu chou industrial technology research institute, chutung, taiwan (republic of china) introduction: size exclusion chromatography (sec) is becoming a widely used technique for separating of extracellular vesicles. various commercially available products were launched on the market, however, their separation efficiencies were not fully disclosed. herein, novel porous agarose microspheres with the tunable diameter and pore size were synthesized by emulsion reaction. the performance was evaluated and compared with commercial products. the modified sec column packing materials were shown to exhibit advantages for rapid, high-recovery and high-purity separation of extracellular vesicles from cell culture-conditioned medium and human plasma. methods: the homemade sec column was packed by gravity flow. 100 μl of the sample was loaded and the pbs buffer was used as eluent. 24 factions were collected and analysed by cd9/cd9 sandwich elisa assay and by micro bca assay for determining respectively extracellular vesicles and total protein content. results: agarose microspheres were prepared by emulsification. the particle size can be controlled by the types and concentrations of surfactants. the product was collected by desired screen meshes and used as packing materials of the sec column. our results showed that the extracellular vesicles were clearly separated from proteins. more than 99.8% of proteins were removed while the recovery of extracellular vesicles was close to 70%, which is much higher than 32% of the commercial product. the total separation time was less than 15 min. summary/conclusion: we have established an approach for generating spherical agarose microspheres as packing materials of homemade sec columns, which are capable of separating extracellular vesicles from complex samples with high efficiency. further validations with additional samples are currently ongoing. immunomagnetic sequential ultrafiltration (isuf) platform for enrichment and purification of extracellular vesicles from large and small volumes of biofluid eduardo reategui, jingjing zhang, luong t. h. nguyen, richard hickey, nicole walters and andre f. palmer the ohio state university, columbus, usa introduction: evs derived from tumour cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. however, compromises have to be made when using a particular technology/methodology for the isolation of evs. currently, there is a trade-off between sample volume and specificity in ev isolation technologies that limits quantitative molecular analysis of ev contents, ultimately impacting the utility of evs in cancer diagnostics. here, we present an approach called immunomagnetic sequential ultrafiltration (isuf). our platform combines ultrafiltration and immunoaffinity separation. using isuf, we demonstrate that small or large volumes of biofluid can be processed (~100 µl or > 100 ml) while concomitantly removing 99.9 % contaminating proteins. we also processed serum from breast cancer patients enabling the characterization of different tumour and immune biomarkers on the isolated evs. methods: human samples were collected under an approved irb. size distribution and concentration of evs were measured using a tunable resistive pulse sensing (trps) method. ev proteins and rnas were extracted and quantified using a bca protein assay and uv spectroscopy. isuf and other ev isolation methods were compared for ev concentration, protein, and rna quantity. results: 50 ml of cell culture media (ccm), 0.5 ml serum, and 100 ml urine samples were processed with the isuf platform and recovered in 100 µl. for all cases, evs were enriched with recovery efficiency greater than 95%. the processing time for a 100 ml sample was 120 min with over 99% of purity. we compared ev concentration and purity isolate from 0.5 ml serum using isuf and other commercially available methods, isuf demonstrated superior performance on isolating evs at high concentrations and purities. analysis of total rna amounts in the isolated evs using different methods was corresponding to higher ev recovery efficiency of isuf. we also compared protein and rna levels of evs enriched with isuf present in urine and serum samples from the same donors (n = 10), and we found that for the same number of evs, the ev rna concentration from both biofluids showed no significant difference. finally, we have processed serum samples from 10 metastatic breast cancer patients and demonstrated that their isolated evs have expression levels of her2, cd24 and mir21 biomarkers at significantly higher levels than healthy controls. summary/conclusion: the isuf platform can be scale-down or -up to work with small or large volumes of biofluids for the isolation of evs. using the isuf platform with clinical samples shows the potential of our platform to be used for cancer diagnosis or monitoring treatment response. funding: national institutes of health (nih) grants ug3tr002884 (e.r.); r01hl 126945, r01hl138116, and r01eb021926 (afp). challenges in exosomes isolation from primary biological samples derived from multiple myeloma patients introduction: multiple myeloma (mm) remains incurable despite advances in its treatment and research progress on the crosstalk between mm and surrounding host cells. exosomes are important regulators of the cellular niche. their importance for diagnostic and therapeutic applications has been proven in many cancers. in this context we hypothesized that a better understanding of the molecular role and features of mm-derived exosomes would provide a basis for their use for both risk stratification and as predictive biomarkers of response to anti-mm drugs already in use in clinical settings, given the optimization and validity of their isolation/purification method. methods: exosomes were isolated from human mm cell lines (hmcls) supernatants and peripheral blood plasma (pbpl) isolated from healthy donors, mm and mgus (monoclonal gammopathy of undetermined significance) patients. both fresh and frozen samples were tested. we evaluated 3 commercially-available kits, density-based separation and ultracentrifugation. results: higher purity and recovery, evaluated by western blotting, nanoparticle tracking analysis and electron microscopy, were observed for supernatant density-based purification and for pbpl resin-based isolation. exploring the function of mm-derived exosomes, we observed an increase in proliferation of the immortalized stromal cell (sc) line hs5 treated with exosomes when compared to untreated cells, and a higher increase in proliferation of scs treated with mm-exosomes when compared to exosomes derived from normal and mgus pbpl samples. summary/conclusion: the method of isolation represents a critical step in the study of exosomes as many factors can affect the purity, yield and downstream application. our data demonstrated that density and resin-based isolation methods provided functional mm-derived exosomes with proliferative effects on scs. altogether our findings may serve as a guide to choose exosome isolation methods for mm studies. further optimization steps, including albumin-depletion from plasma samples and use/type of serum in cell cultures, should be taken into consideration when planning proteomics and genomics as downstream applications. funding: australian government rtp and monash departmental scholarship. a rigorous method for exosome isolation from post-mortem eyes introduction: in order to determine and validate the tissue-specific content of extracellular vesicles (evs) in biofluids, robust ev isolation methods from tissues must be developed. however, to date very few rigorous methods to isolate or enrich for intact evs from tissues have been reported. we present a comprehensive exosome isolation method with a sufficient level of characterization to unequivocally demonstrate true ev identity from ex vivo eyes. methods: iodixanol (optiprep) buoyant density gradient ultracentrifugation (dguc), cushioned dguc (c-dguc), and our newly developed c-dguc immunocapture (c-dguc-ip) method were used to compare yield and enrichment of exosomes isolated from porcine eyes between 3 to 5 hours post-mortem. yield was assessed by nanoparticle tracking analysis (nta) and immunoblotting for exosomal markers along with total protein quantitation. enrichment was assessed by comparison of exosomal markers, ocular-specific markers and known contaminant markers, plus in-depth proteomic mass spectrometry analyses. results: high enrichment of posterior eyecup small evs (sev) were achieved by dguc and c-dguc, with c-dguc resulting in an eightfold increase in yield by nta and two to fivefold increases of exosomal protein markers such as syntenin-1 and cd81 by immuno-blotting compared to dguc. interestingly, in-depth proteomic analyses revealed that a majority of these sevs with densities of 1.07-1.11 g/ml isolated by dguc and c-dguc were likely of endoplasmic reticulum (er) and golgi origin, suggesting er-to-golgi transport vesicles resulting from post-mortem tissue cell rupture. in order to enrich further for sevs (including exosomes) we subjected sevs isolated by c-dguc to anti-cd81 immunocapture. the resulting sev proteome was enriched 1.5-to 4-fold for bona fide sev and exosome markers compared to c-dguc. summary/conclusion: the c-dguc method provides an enhanced yield and purity of sevs and exosomes from ex vivo eye tissue. however, to avoid significant contamination with er and golgi-derived vesicles from postmortem eyes, a final ev-specific immunocapture step is required to achieve sufficient purity for subsequent analyses. our highly rigorous method paves the way for identification and validation of ocular-derived exosomes in blood and their potential use as eye disease biomarkers. characterization of the extraction of extracellular vesicles using a lab-ona-disc filtration system introduction: personalized treatment for cancer is a promising way to face the multiplicity of the disease, to increase the efficacy of drugs and to decrease their toxicity. as part of this strategy, liquid biopsy explores a new non-invasive approach to diagnose cancer, guide treatment and monitor its efficacy. extracellular vesicles (evs) are nanometric lipid bilayers micelles with high potential as biomarkers. they are involved in the transfer of information (proteins, rna and dna) between cells. evs include a broad spectrum of particle sizes, from the tens to thousands of nanometres. the isolation of evs from complex matrices is the first step of any protocol and is particularly important for the reproducibility and fidelity of the results presented, as it could bring bias in further analysis. in order to explore the heterogeneity of evs, a full characterization (physical and biological) of the extracted evs is needed. we evaluate and compare 3 evs purification methods, including ultracentrifugation, sizeexclusion chromatography (sec) column and an emerging microfluidic technology: labspinner filtration labon-a-disc device isolating evs between two filters of 600 and 20nm. methods: a431 cell supernatant was used as a model matrix. we compared three methods of extraction of evs: ultracentrifugation with two cycles of 1 h10 at 110,000 g at 4 degrees celsius (rotor type 70ti, beckman floor ultracentrifuge optima l90 k), qev size exclusion chromatography columns from izon (qevoriginal/70 nm) and lab-on-a-disc filtration system (labspinner, exodisc c). evs characterization was conducted with nta (nanosightns500), trps (izon), nanodrop (spectrometernd1000), tem (fei tecnai 12 120 kv) and custom micro-immuno-assay. results: in this study, we characterize a filtration system made of two serial filters of 600 nm and 20 nm pores for isolation of evs. compared to ultracentrifugation and chromatography columns, yield of extraction is up to 10 times higher and the size of the extracted particles is smaller. tem imaging was used for assessment of the quality of the extracted evs. however, albumin concentration measurement tends to show that the purity of the solution is decreased. the immuno-labelling analysis shows that the proteomic signature of the extracted evs differs according to the extraction methods. the new filtration technology seems to give us access to a broader range of evs compared to standard methods. summary/conclusion: in this study, we characterized 3 purification methods including lab-on-a-disc filtration, and were able to demonstrate an increase of the concentration of evs by a factor of 10, a decrease of the size of the accessible extracted particles and access to new proteomic signatures. funding: we acknowledge the support of génome québec and action marie skłodowska-curie. effects of sample processing on isolation of extracellular vesicles from blood plasma by centrifugation darja božič a , matej hočevar b , veno kononenko c , marko jeran a , urška štibler d , immacolata fiume e , manca pajnič f , ljubiša pađen f , ksenija kogej g , damjana drobne c , ales iglič h , gabriella pocsfalvi i , veronika kralj-iglič f and darja bozic j introduction: the isolation of extracellular vesicles (ev) from body fluids is still controversial and the poor understanding of vesicle stability and effects of sample processing is probably one of the core issues preventing the breakthrough of this field into applicative practices. methods: we performed an in-depth study of sample changes in blood, blood plasma and samples throughout the increasing speed of centrifugation, considering the number, size, contents and shape of particles in the isolates. flow cytometry, light scattering, mass spectrometry and scanning electron microscopy were employed to reveal the properties of material in the samples. results: the particles of size about 100-500 nm with characteristic topology of membrane vesicles without internal structure were observed by the scanning electron microscope only in ev isolates prepared from fresh blood sample. inspection of the tube surface in which the isolation took place suggests that those particles are likely formed from activated platelets tearing at the tube wall due to the centrifugal pull. the isolates prepared from frozen blood plasma prepared by centrifugation with different forces contained different amounts of particles with similar protein contents, predominated by highly abundant human plasma proteins, including albumins and immunoglobulins. some lipoprotein clearance and fibronectin precipitation were however observed through increased speed and time of centrifugation. summary/conclusion: the results of this study [1] contribute to the understanding of stability and dynamics of membrane particles. the reported evidence provides the support for viewing ev isolates as a product, shaped by uniqueness of the starting samples and the thermal and mechanical stress applied upon processing. we believe this kind of insights strengthen our ability of reading the story of evs. introduction: apoptosis is a form of programmed cell death with diverse roles in the tumour microenvironment and emerging data show that, besides its role in tumour suppression, it can also promote oncogenic proliferation. highly aggressive tumours such as burkitt lymphoma (bl) show high levels of apoptosis, which has a diagnostic and prognostic value for classifying and staging the disease. we hypothesize that amongst other elements, extracellular vesicles (ev) are key mediators of apoptotic cell-derived tumour microenvironment signals. here, we report on ev released in vitro by apoptotic bl cells (apo-ev) in relation to their potential use as cancer biomarkers. methods: basic physical properties of apo-ev such as structure, size distribution, surface charge and membrane fluidity are discussed using cryo electron microscopy (em) and tomography, nanoparticle tracking analysis, dynamic light scattering and fluorescence anisotropy respectively. for phenotypic analysis we apply immunocapture and flow cytometry, immunogold labelling on transmission em, fluorescence microscopy and quantitative pcr. in addition, we study the interaction of apo-ev with blood components such as platelets, leucocytes and red cells, in order to understand their effects in the circulation and therefore their potential for analysis in blood samples. results: looking at the differences between apo-and non-apo-ev, apo-ev have larger diameter, while structurally are not different. however, we have identified distinct apo-ev markers such as active caspase 3 and histones, or dna and small non-coding rna-y. there is also strong interaction of ev with platelets and leucocytes but not with red cells, indicating potential routes of transfer of ev cargo in the circulation. summary/conclusion: it is concluded that for the characterization of the heterogenous ev populations, combination of multiple techniques is often required, and also, understanding the strengths and limitations of each method is essential for choosing the appropriate set of analytical tools. finally, we consider that monitoring free circulating apo-ev or blood cells with which they have interacted is a promising approach to improve cancer diagnosis, prognosis and evaluation of therapeutic response. casting a small netrin: functional roles of a novel surface factor on stroma-derived extracellular vesicles in pancreatic cancer kristopher s. raghavan a , ralph francescone b , janusz franco-barraza b and edna cuckierman b a drexel university; fox chase cancer center, philadelphia, usa; b fox chase cancer center, philadelphia, usa introduction: pancreatic ductal adenocarcinoma (pdac) is a devastating disease driven and supported by changes in its microenvironment, or stroma. here we dissect the intercellular communication that exists between the primary stromal component, cancer-associated fibroblasts (cafs) and pdac. pdac communicates with its microenvironment, in part, through the exchange of specific types of extracellular vesicles (evs). specifically, we focus on the mechanism by which caf-secreted evs support pdac survival, with an additional goal to identify biomarkers suitable to generate a future "liquid biopsy" test for early pdac detection and prognosis. methods: evs are isolated from patient-derived pdac-associated fibroblasts via differential ultracentrifugation and validated by isev standards. human pdac cell lines used as recipient cells are treated with caf-evs to assess their role in supporting pdac survival. recombinant proteins, neutralizing peptides, and non-functional mutant proteins are used to block ev interaction with target cells. results: we observe sub-types of caf-evs containing unique surface receptors. one ev sub-population of interest contains a novel surface protein (nsp) expressed on the plasma membrane of pancreatic cafs, but not their healthy counterparts. further, pdac cells up-regulate nsp's lone binding partner, suggesting a role for these factors in pdac-selective ev uptake. functional assays designed to test pdac viability suggest these nsp(+)-evs protect pdac cells from programmed cell death as a result of physiological stress. this ev-mediated survival benefit can also be inhibited by blocking the interaction of nsp and its binding partner, suggesting the engagement of these two factors is necessary for cafs to support pdac via evs. pursuing our biomarker goal we confirm stromal nsp expression increases during early panin stages prior to tumour development, and we are currently seeking to validate nsp(+)-evs in blood of pdac patients. summary/conclusion: this research shines light on a novel mechanism of tumour-stroma communication that may be crucial for cancer progression during early disease stages and a potential target for disrupting the supportive role of the tumour microenvironment. additionally, we describe a sub-population of nsp (+)-evs that have the potential to serve as biomarkers for identifying pdac development. exosomes carry distinct mirnas that drive medulloblastoma progression introduction: extracellular vesicles (evs) represent an ideal source of functional biomarkers due to their role in intercellular communication and their ability to protect cargo, including rna, from degradation. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the bloodbrain-barrier. here we characterised the rna of exosomes isolated from medulloblastoma cell lines, with the aim of investigating exosomal rna cargo as potential functional biomarkers for medulloblastoma. methods: exosomes derived from a panel of matched (original tumour and metastasis) medulloblastoma cell lines were isolated and characterised by nanosight, electron microscopy, western blotting and nanoscale flow cytometry. exosomal mirna and mrna from our matched cell lines and foetal neuronal stem cells, which were used as a normal control, were analysed by rna-sequencing technology. results: based on hierarchical clustering, malignant derived exosomes were distinctly separated from normal control exosomes. mirna profiling revealed several established oncomirs identified in our malignant derived exosomes compared to control samples. using interaction pathway analysis, we identified that our malignant exosomes carry numerous mirnas implicated in migration, proliferation, cellular adhesion and tumour growth. several previously identified oncomirs were also identified to be present at higher levels in metastatic exosomes compared to primary and normal, including hsa-mir-455-3p and hsa-mir-92a-3p. summary/conclusion: this study shows that exosomes from mb cells carry a distinct mirna cargo which could enhance medulloblastoma progression. the use of circulating exosomes as markers of metastatic disease could be an innovative and powerful noninvasive tool. introduction: inflammatory changes in the bone marrow (bm) and suppression of haematopoietic stem and progenitor cell (hspc) function during acute myeloid leukaemia (aml) significantly contribute to patient morbidity and mortality. our laboratory has previously shown that aml-derived extracellular vesicle (ev-aml) trafficking confers a state of enforced quiescence and leads to lasting dna damage in hspcs. here we explore the underlying cause. specifically, we hypothesize that ev-aml incite inflammatory regulators as potential mediators of dna damage. methods: as a validated model of aml, we utilized the murine tib49 cell line as a source of ev-aml. ev-previous work has indicated that mirnas, notably mir-146a and mir-155, play a critical role in scc tumour development. evs are membrane-bound vesicles involved in cell-cell communication carrying actively sorted cargo, protected from degradation. the potential pathways these vesicular mirnas modulate and the implication they have on cancer biology is under active investigation. we have previously shown that the cadherin dsg2, a stem cell marker, modulates ev release. dsg2 is upregulated in a number of cancers, including scc, and correlates with poor prognosis. here we aim to elucidate the impact of ev-associated mirnas in sccs by bioinformatic analysis. methods: scc cells stably expressing dsg2 were generated and evs isolated by sequential ultracentrifugation. total cellular and ev rna was isolated by mirneasy, analysed using rnaseq and identified by grch37 alignment. results were confirmed by qpcr. altered pathways based on targets were identified using mirnet and kegg pathway analysis. potential cancerassociated cytokine targets were confirmed by antibody array. results: rnaseq revealed 87 cellular and 15 ev mirnas that were differentially expressed in response to dsg2 with 7 overlapping. the highest altered mirnas were validated by qpcr. kegg pathway analysis determined that these mirnas have the highest number of shared targets in cancer, cell cycle, and p53 signalling pathways. interestingly, mir-155 was upregulated while mir-146a was dramatically downregulated in evs. targets of mir-146a, icam-1, il-6, and il-8, cytokines critical for cancer progression were upregulated. summary/conclusion: these results suggest that the mirna content of evs is tightly regulated. by altering the mirna profile, dsg2 contributes to the pathogenicity of these evs by increasing levels of cytokines important for cancer stem cell renewal and metastasis. in addition, these mirnas may serve as non-invasive diagnostic markers for sccs. funding: nih r01 cancer cells grown in 3d release distinct extracellular vesicles during tumour growth and invasion jens c. luoto, sara bengs, leila coelho rato, lea sistonen and eva henriksson åbo akademi university, turku, finland introduction: cancer cells secrete extracellular vesicles (evs) that affect tumour progression. the characteristics of evs produced during tumour growth and invasion are however poorly understood. in this study, we identify the composition and characteristics of evs produced by noninvasive and invasive tumours and correlate these characteristics with the invasive status of the tumour. for that purpose, we established a protocol for isolating evs from extracellular matrix (ecm)-based three-dimensional (3d) cancer cell cultures. methods: human prostate cancer pc3 cells were grown in 3d cultures using ecm-based hydrogel, in standard 2d culture conditions and in bioreactor. evs were isolated from these cultures with differential and density gradient centrifugation. the isolated evs were characterized with nanoparticle tracking analyses, electron microscopy, immunoblotting and mass spectrometry (ms). results: our results demonstrate that 3d ecm-based hydrogel cell cultures secrete evs that can be isolated from both the conditioned media and the hydrogel. the invasive 3d cultivated pc3 organoids were found to secrete large amounts of evs compared to the non-invasive organoids. interestingly, our ms results revealed that non-invasive and invasive organoids secrete evs with partially distinct protein cargo. summary/conclusion: we have established a novel protocol for ev production in a 3d cell culture system utilizing ecm-based hydrogel, in which invasive tumour growth can be mimicked. our method allows the specific isolation and characterization of evs derived from different stages of 3d culture, such as non-invasive and invasive organoids. importantly, we found that tumour-derived evs change in composition during the tumour progression. taken together, our method can be used to define the distinct ev characteristics involved in cancer invasion. we previously showed extracellular vesicles (evs) to be causally involved in transmitting drug resistance. this study aimed to evaluate compounds proposed to reduce/block ev release. specifically, we selected calpeptin and y2763 (proposed to inhibit evs budding from the cell membrane) and manumycin a and gw4869 (proposed to inhibit evs deriving from mvbs). associated effects on -and consequences of-ev release were then investigated. methods: suitable compounds concentrations that were non-toxic to cells were first selected by performing cytotoxicity assay and flow cytometry (fc). conditioned medium (cm) was collected from docetaxel-resistant pc3 (pc3 rd) cells after 48 h incubation in dfbs-medium with or without the 4 compounds. evs were separated from tangential flow filtration concentrated cm using optiprep density gradient. 1.03-1.16 g/ml fractions were then pooled and washed. evs were characterised using nta, immunoblot, tem and lipid assay and fc. influences on growth and migration, of evs continuing to be released (at 1x105evs/3x103cells, 1x107evs/3x103cells), were evaluated on recipient du145 and 22rv1 cells. evtrack id ev190113, score 88% results: calpeptin and y27632, alone and in combination, did not significantly affect quantities of evs released. however, gw4869 significantly (p < 0.004) increased quantities of released evs, of a larger size; very high protein to lipid ratio; and carrying grp94 compared to control evs (p < 0.006). this effect was reverted when gw4869 was combined with manumycin a (p < 0.004). following all compounds treatments, 1x105evs/ 3x103cells inhibited 22 rv1 proliferation (p < 0.0014), while at 1x107evs/3x103cells only evs from manumycin a (p < 0.05) and y27632 (p < 0.00036) treated cells reduce 22 rv1 proliferation. evs following gw4869 treatment significantly (p < 0.001) inhibited du145 migration compared to bulk non-treated control and compared to the effect obtained using the entire pool of evs (p < 0.001). summary/conclusion: while none of the 4 proposed inhibitors significantly reduced ev release, the resulting evs were less potent in transmitting aggressive behaviour, such as proliferation and migration, to receiving cell lines. patient-derived organoids represent a novel tool to study the effect of intra-tumoral heterogeneity on ev release in non-small cell lung cancer introduction: lung adenocarcinoma (luad) is the leading cause of cancer-related death with a low 5-year survival. although the importance of intra-tumoral cellular heterogeneity of solid tumors in the clinical outcome and treatment is emerging, proper models to study its effects on ev release and cargo in human tissues still lack. the 3d organoid technology maintains the cellular and genetic heterogeneity of in vivo tissues and has proved to be so far the best ex vivo model of human cancers. by using patient-derived and mouse organoids we set out i) to compare the ev release from normal and tumor tissues and ii) to follow changes in ev secretion when the relative ratio of tumor cell subpopulations is shifted. methods: we used mouse and luad patient-derived normal and tumor organoids. the medical research council of hungary approved our experiments with human samples and informed consent was obtained from patients. evs were detected by antibody-coated beads, nta and tem. intra-organoid heterogeneity was proved by immunostaining and rt-qpcr. results: we provide evidence that both mouse and human normal organoids contain all the bronchiolar cell types. interestingly, luad organoids selected for tp53 mutation contained not only ki67+ proliferating cells, but differentiated cell types as well. furthermore, all the lung organoid cultures produced evs and this was shifted to the smaller size range. interestingly however, when modifying the proportion of organoid cell types, we observed an increased ev release when more ki67+ proliferating cells were present both in normal and in luad samples. summary/conclusion: our data show that patientderived lung organoids represent a novel model to study the role of intra-tissue heterogeneity in ev functions in the humans, leading to improved diagnosis. funding: this work was funded by the national competitiveness and excellence program nvkp_16-0007 (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). exosome mediate heart-adipocyte communication after myocardial ischaemia/reperfusion and impairs adipocyte endocrine function yajing wang, lu gan, dina xie, wayne lau, theodore christopher, bernard lopez and xinliang ma thomas jefferson university, philadelphia, usa introduction: by incompletely understood mechanisms, mi patients sustain systemic metabolic disorder. adipocytes are an important cellular type regulating energy homoeostasis. the impact of mi upon adipocyte function remains unknown. exosomes (exo) are critical vehicles mediating organ-organ communication. however, whether and how exo may mediate post-mi cardiomyocyte/ adipocyte communication have not been previously investigated. methods: adult male mice were subjected to mi/r. serum exo were isolated 3 hours after r and incubated with 3t3 l cells for 24 hours. the effects of exo upon adipocyte function were determined. results: compared to control, mi/r exo significantly altered the expression of 17 genes known to be important in adipocyte function. go analysis revealed that genes associated with endoplasmic reticulum (er) function and adipocyte endocrine function are the primary two pathways altered by mi/r exo. venn analysis identified 11 mi-rnas as cardiac-enriched, adipocyte-poor, and er function-related mirnas. rt-qpcr confirmed the mir-23a/27a/24-2 family members are the most markedly increased mi-rnas in mi/r exo. incubation of 3t3 l cells with mi-r27a mimic significantly downregulated edem3, dsba-l, and pparn, and upregulated perk and chop. conversely, mi-r27a inhibitor significantly decreased the impact of mi/r exo upon er function genes. additional studies demonstrated edem3 and pparγ (two critical molecules maintaining er function and adipocyte endocrine function) to be direct targets of mi-r27a. one of the most significant endocrine molecules of adipocyte origin, adiponectin is regulated by pparn at the transcriptional level and by dsba-l at the post-translational level. we next determined whether mi/r exo may affect adiponectin expression/ assembly. incubation of 3t3 l cells with mi/r exo significantly inhibited total and high molecular weight adiponectin expression, an effect blocked by mir27a mimic. finally, in vivo administration of gw4869 (exo biogenesis inhibitor) or mir27a inhibitor attenuated adipocyte er dysfunction and restored plasma adiponectin level in mi/r animals. summary/conclusion: we demonstrate for the first time that mi/r causes significant adipocyte er and endocrine dysfunction by exo mediated cardiomyocyte/adipocyte communication via mir-23a/27a/ 24-2. funding: nih and american diabetes association pancreatic cancer cell extracellular vesicles drastically alter the behaviour of recipient normal pancreas cells charles p. hinzman a , yaoxiang li a , meth jayatilake a , jose trevino b , partha banerjee a and amrita cheema a a georgetown university medical center, washington, usa; b university of florida health science center, gainesville, usa introduction: pancreatic cancer (paca) is predicted to become the 3rd leading cause of cancer-related deaths by 2030. patients diagnosed with pancreatic ductal adenocarcinoma (pdac) have a 5-year survival ratẽ 9%. detection of pre-neoplastic lesions can potentially improve survival. however, there is currently no screening test for early stage detection. importantly, paca tumours are 90% non-tumorigenic cells. a better understanding of early paca oncogenesis is needed. cancer cells shed extracellular vesicles (evs) that are internalized by neighbouring and distant cells to induce a myriad of cancer progression events. we hypothesize that in early paca oncogenesis, evs mediate a behavioural change in surrounding normal cells, leading to the formation of this unique stroma. the purpose of this study was to develop a model to examine the phenotypic changes undergone by normal human pancreas cells when they are exposed to paca cell evs. methods: evs were isolated using differential ultracentrifugation with filtration from established (panc-1, sw-1990, capan-1 and miapaca-2) and patientderived xenograft (ppcl-46 and ppcl-68) paca cell lines. cells were grown using ev-depleted fbs. ev isolations were validated and quantified using transmission electron microscopy, quantitative elisa, immunoblot and nanoparticle tracking analysis. normal pancreas cells (htert-hpne and hpde-h6c7) were co-cultured with cancer cell evs for 24-48 hours. metabolic activity was measured using a mito stress test on a seahorse xfe96 extracellular flux analyser. results: we discovered that normal cells undergo vast behavioural transformations, including significant morphological changes, increased proliferation and an uncharacteristic invasive capability, when co-cultured with paca cell evs. these responses were ev dose dependent. further, paca cell evs metabolically reprogrammed normal cells, causing a bioenergetic switch, from a quiescent, aerobic profile to a highly energetic and glycolytic profile. summary/conclusion: our results indicate that paca cell evs confer enormous transformational properties to normal human pancreas cells in vitro. we hypothesize that evs impart distinct transformational properties to normal cells in vivo and this influence could unveil novel mechanisms regulating cancer onset and progression. these signals may be detectable before progression of early-stage paca to pdac, leading to the development of assays for earlier diagnosis in patients. further studies are underway to identify the biochemical mediators of these changes. plasma extracellular vesicles-mirnas released by hypoxic cells are associated to pro-tumorigenic and immunosuppressive microenvironment in lung cancer introduction: extracellular vesicles (ev) containing specific subset of functional biomolecules, such as micrornas (mirnas) are released by all cell types. it has been widely demonstrated that ev-mirnas from cancer cells can manipulate the tumour microenvironment modulating the gene expression of recipient cells. we previously identified a three levels risk classifier (msc) based on 24 plasma-mirnas associated with lung cancer development and prognosis. the aim of this study was to investigate the potential role of ev-24 mirnas as mediators of pro-tumorigenic features. methods: evs were isolated from plasma of heavysmoker individuals with high (mscpos) or low (mscneg) risk of lung cancer by differential centrifugation method. purity of evs isolated was confirmed by sizing using nta and tem analysis and expression of ev-enriched proteins. mirna levels were analysed by dpcr. in vitro and in vivo analyses were used to assess the biological effect of plasma evs on different recipient cells. results: levels of mirnas in evs correlated with determination of whole plasma (80% of correlation with msc classifier). mscpos-evs stimulated 2d and 3d proliferation of non-tumorigenic epithelial cells through c-myc transfer into recipient cells. furthermore, mscpos-evs increased the ability of huvec to form tubular structures compared to mscneg-evs. in vivo co-injection of mscpos-evstreated huvec with a549 lung cancer cells resulted in an increase of tumour growth compared to mscneg-evs-treated huvec. mir-126 modulation in evs with mirna mimics or in recipient cells using mirna inhibitors demonstrated that this mirna is implicated in the autocrine proangiogenic modulation of huvec phenotype. mscpos-evs induced m2 polarization of macrophages both in vitro and in vivo. we demonstrated using synthetic oligonucleotides that mir-320 is responsible for the immunosuppressive modulation of these cells. regarding the potential origin of evs in mscpos individuals, we observed that hypoxia stimulated the secretion of evs containing c-myc from fibroblasts, mir-126-evs from endothelial cells and mir-320-evs from granulocytes. summary/conclusion: these data show that plasma evs of high-risk individuals display pro-tumorigenic features, as documented by their ability to induce a pro-angiogenic and immunosuppressive microenvironment suggesting an involvement of evs in lung cancer development. exploration of ev-associated metastatic targets on melanoma cells introduction: cancer cells secrete evs that may harbour metastatic proteins. previous studies have demonstrated the decrease of cd63 tetraspanin expression in melanoma cells are correlated with enhancing its metastatic potential. however, other proteins, such as cd44, cd47, met and nrp2 which are overexpressed in melanoma cells, are also associated with the spread of cancer. in this study, we sought to investigate the expression of metastatic proteins in evs derived from murine melanoma b16f10 lineage. methods: b16f10 cells were cultured in dmem supplemented with 10% fbs and antibiotics. the cells supernatant were harvested each 24 hours, filtered through 0.22 µm and ultracentrifuged at 110000 x g for 90 min at 4ºc to pellet evs. next, size and concentration was determined using nanoparticle tracking analyses technique, and the morphology of evs were analysed by negative-staining transmission electron microscopy (tem). the ev's surface protein were characterized by flow cytometry and protein content was profiled by mass spectrometry. results: our flow cytometry results have shown the presence of tetraspanins markers cd63, cd9 and cd81 on vesicle´s surface. in addition, our assay demonstrated a diminished expression of cd63 molecule in comparison to cd9 and cd81. we have profiled melanoma-evs by mass spectrometry, identifying the presence of proteins that may be associated to metastasis, such as cd44, cd47, met and nrp2. summary/conclusion: these preliminary results are consistent with the literature and suggest that melanoma-derived evs harbour proteins, which may contribute to promote tumour metastasis. in our next step, we plan to generate b16f10 lineages harbouring shrna vectors, in order to knockdown gene expression of cd63, cd44, cd47, met and nrp2 to investigate the metastatic potential in vitro, in comparison to parental cells. we also may use syngeneic mice models to explore metastatic potential of genetically modified b16 f10-derived cells. introduction: overexpression of her2 occurs in2 0% of breast cancers and confers aggressive behaviour and poorer prognosis. thankfully, a number of drugs such as neratinib have been developed to target her2, potentially providing substantial benefit for many patients. nevertheless, it is estimated that up to 70% of patients with her2-overexpressing tumours do not gain benefit, as a result of innate or acquired drug-resistance. this study aimed to investigate if nano-sized membrane-surrounded extracellular vesicles (evs) released from drug-sensitive and drug-resistant cells reflect the her2 status of their cells of origin and thus have potential as minimallyinvasive biomarkers. methods: evs were isolated from conditioned media (cm) of her2-positive cell lines (hcc1954 and skbr3) and their neratinib-resistant counterparts (hcc1954-nr and skbr3-nr) that we developed in our laboratory. evs from cm of a triple-negative breast cancer (tnbc) cell line variant, hs578 ts(i)8, were evaluated as negative control for her2. in brief, cm was centrifuged at 300 g, for 10 min x3 to get rid of any cells. the supernatant was then centrifuged at 200,000 g for 6 h at 4ºc to collect evs. the resulting evs were washed in pbs, centrifuged as before, and resuspended in 100 μl pbs. ev quantities were estimated by nanoparticle tracking analysis (nts). ev lysates were characterised by immunoblots, for established positive and negative ev markers. particle concentration as well as size distribution of evs were measured using the zetaview (particle metrix, germany). surface proteins on single evs were analysed by highresolution flow cytometry (fc), using an amnis imagestreamx mark ii. all data was submitted to ev-track (ev-track id: ev190112). results: neratinib-resistant cell line variants were found to have reduced her2 protein expression compared to their respective drug-sensitive counterparts. neratinib-resistant cell line variants released fewer evs, when normalised per number of secreting cells, compared to their-drug sensitive counterparts. furthermore, evs from drug-sensitive cells carried her2, while those from drug-resistant cells lacked her2 (similar to the evs from the tnbc cells); reflecting the her2 status of their cells of origin. summary/conclusion: this study indicates that a reduction in her2 protein expression is a mechanism by which cancer cells manifest resistance to her2-targeted drugs (i.e. by making fewer her2 receptors available on the cells surface to accommodate the drugs activity). furthermore, ev-carried her2 seems to reflect the her2 status of their cells of origin. this suggest that analysis of her2 on evs in the peripheral circulation may help predict response to her2-targeted drugs. thus, analysis of evs in appropriate cohorts of patients' specimens is warranted. introduction: rab27a, a small gtpase involved in exosome biogenesis by regulating mve docking at the plasma membrane, and its expression level highly correlated with ohsv replication ability in vitro. oncolytic viruses is a newly promising therapeutic agent for cancer treatment. however, more than 50% of tumours naturally showed highly resistant to oncolytic viruses for unknown reasons. uncovering the underlying mechanisms of resistance to ohsv can offer potential therapeutic targets to enhance ohsv activity to kill tumour cells. in addition, it will give new insights into the identification of therapeutic biomarkers, which can be used to predict patient response to ohsv in clinical. methods: deep-sequencing data showed that lower expression level of rab27a is present in ohsv resistant tumour cells compared to that in sensitive tumour cells. then an ohsv resistant mc38 tumour cell line was established by repeated injections with ohsv in mc38 tumour-bear mouse model. lastly, it was verified that ohsv resistance is associated with a downregulation of rab27a and overexpression of rab27a can rescue ohsv replication. results: 1) the lower expression level of rab27a is shown in ohsv resistant tumour cells compared to that in sensitive tumour cells shows in deep-sequencing data. 2) furthermore, we established an ohsv resistant mc38 tumour cell line by repeated injections with ohsv in mc38 tumour-bear mouse model. similarly, in ohsv resistant mc38 cell line, rab27a expression was lower than parental mc38 cells. and the release of exosomes and virus was decreased in ohsv resistant mc38 cell line. these results were confirmed by rab27a sirna knockdown. 3) we verified that in ohsv naturally resistant human cancer cell lines, ohsv resistance is associated with a downregulation of rab27a and overexpression of rab27a can rescue ohsv replication. summary/conclusion: downregulation of rab27a expression restricts the efficiency of ohsv replication and the spreading ability of the released progeny virus which also provide rab27a as a potential target and biomarker for ohsv cancer therapy. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd2015071414385495, shenzhen science and innovation commission project grants jcyj201 70411094933148, jcyj20180306173333907 to shenzhen international institute for biomedical research. inactivation of emilin-1 by proteolysis and secretion in extracellular vesicles favours melanoma progression and metastasis introduction: studies have demonstrated that melanoma-derived extracellular vesicles (evs) home in distal organs and sentinel lymph nodes favouring metastasis. although lymph node metastases are themselves rarely life threatening, they could be considered as one of the first step of metastasis in many cancer types. therefore, defining the mechanisms involved in lymph node metastasis and pre-metastatic niche formation in lymph nodes could bring the clue to block the metastatic process from the beginning. methods: we have characterized secreted exosomes from a panel of mouse melanoma models representative of low metastatic potential (b16-f1), high metastatic potential (b16-f10) and lymph node metastasis (b16-f1 r2). we analysed the rna expression in cells and protein content of exosomes derived from mouse melanoma lymph node metastatic models by rna sequencing and mass spectrometry respectively. we validated expression by western-blot, qpcr and immunofluorescence. to define the mechanism of emilin-1 secretion cells were treated with the ev secretion inhibitor (non-competitive inhibitor of sphingomyelinase), gw4869. cell viability and cell cycle assays with an overexpression model (b16-f1-emilin-1) were also performed. in vivo experiments based on subcutaneous and intrafootpad injection for studying the role of this protein during melanoma progression were performed to define the relevance of our findings in vivo. human paraffin samples were analysed for emilin-1 expression by immunohistochemistry. results: we found a signature of over-expressed genes and hyper-secreted proteins in exosomes related to lymph node metastasis in the b16 mouse melanoma model. among them, we found that emilin-1, a protein with an important function in lymph node physiology, was hyper-secreted in exosomes. interestingly, we found that emilin-1 is degraded and secreted in exosomes as a mechanism favouring metastasis. further, we found that emilin-1 has a tumour suppressive-like role regulating negatively cell migration. importantly, our in vivo studies demonstrate that emilin-1 overexpression reduced primary tumour growth and metastasis in mouse melanoma models. analysis in human melanoma samples showed that cells expressing high levels of emilin-1 are reduced in metastatic lesions. summary/conclusion: overall, our analysis suggests that the inactivation of emilin-1 by proteolysis and secretion in exosomes reduce its intrinsic tumour suppressive activities in melanoma favouring tumour progression and metastasis. funding: this work was supported by grants from mineco (saf2014 54541 r), asociación española contra el cáncer, fundación de investigación oncológica fero and mineco-severo ochoa predoctoral program. introduction: the amplification of erbb2 gene and the consequent overexpression of the encoded protein her2 have an important role in breast cancer classification at diagnosis and subsequent treatment decision with the anti-her2 monoclonal antibody trastuzumab. fish and ihc have been used so far to detect erbb2 gene amplification and her2 protein overexpression respectively in tissue biopsies. in this context, a major goal for liquid biopsies is to take advantage of the information carried by circulating tumourderived materials (such as extracellular vesicles (evs) and cell free dna (cfdna)) to noninvasively detect erbb2 gene status in the blood. however, the isolation of diverse tumour-derived materials from a single aliquot of patients' plasma and the accurate detection of cancer biomarkers is still challenging. methods: by adopting a recently published nickelbased evs isolation (nbi) protocol1 that allows for recovery of cfdna after evs isolation, we generated a high-sensitivity molecular assay to accurately detect erbb2 amplification and consequent her2 overexpression on a limited volume of plasma (1.5 ml) collected from breast cancer patients (stage i, ii and iii) at diagnosis. results: 1) we detected erbb2 amplifications by droplet digital pcr (ddpcr) on the cfdna isolated from the plasma of erbb2 positive patients; 2) we confirmed her2 overexpression on a subset of patients by setting up an antibody-based affinity reaction designed to detect her2 protein on the surface of the isolated evs; 3) we succeeded in the quantification of her2 transcripts enclosed within evs by performing ddpcr in samples of patients showing a range of circulating tumourderived material. the specificity and sensitivity of these novel methodological assays were tested on a cohort of healthy individuals (n = 20) and on a cohort of her2 positive (n = 20) or her2 negative breast cancer patients (tnbc; n = 20). summary/conclusion: here we report a pilot study on a novel multimodal method for erbb2 detection from a minimal amount of plasma. this approach integrates information from cfdna with evs-derived rna and proteins analysis. this proof of concept may ultimately translate into relevant clinical applications for disease diagnosis as well as for therapy selection and monitoring of disease progression. introduction: the biological and medical importance of exosomes recognized over the last decade has given rise to a crucial need for the discrimination between true evs and co-purified nanoparticles, such as lipoproteins, protein aggregates or debris. additionally, it is imperative to develop methods to identify and characterize ev sub-populations. considering ev biology and the reliability of labelled biomolecules, we developed both exogenous and endogenous labelling protocols, taking advantage of different dye properties which can target a multitude of compartments. this approach reveals key aspects of ev structure and integrity. methods: nanosized evs/exosomes were purified by size exclusion chromatography (sec) from model cell lines and human plasma. diverse dyes were orthogonally evaluated through different single particle and bulk analysis technologies, such as high-resolution cytometry, nanoparticle tracking analysis and plate fluorimeter. concomitant profiling of specific ev subpopulations was optimized using antibodies (abs) against tetraspanins and cell type specific targets and assessed by single particle analysis and elisa. to assess specificity of labelling protocols we used specific controls such as recombinant rfp expressing vesicles and purified lipoproteins. results: our ev staining protocols allowed for high labelling efficiency and unprecedent ev discrimination, quantification and characterization by combining single particle analysis and bulk measurements in simple matrices (saline buffers) and in complex biofluids (i.e. plasma). different approaches have diverse and complementary advantages (costs, capacity, sensitivity, informative readout) for implementation in research and diagnostic development flows, directly feeding in-house r&d and qc pipeline. summary/conclusion: overall, fluorescent evs are versatile and valuable tracers that can be applied in the optimization of pre-analytical and purification protocols, selection of target biomarkers and diagnostic assay calibration and validation. funding: endevor (por-fse 2014-2020) region tuscany and exosomics r&d program. subpopulations of tissue-derived extracellular vesiclesmethodological evaluation for vesicle size measurement introduction: introduction: subpopulations of extracellular vesicle (evs) are commonly classified by their different size, however, the ev size cut off is still under discussion. the aim of the study was to evaluate size range of six ev subpopulations using three methods: electron microscopy (em), nanoparticle tracking analysis (nta) and exoview™. methods: methods: ev subpopulations were isolated from melanoma tissues by a centrifugation based protocol recently established in our lab. large and small evs (levs and sevs) were isolated with differential ultracentrifugation and these were further separated into low and high-density fractions (ld and hd). size of ev subpopulations was then evaluated by: em introduction: small-extracellular vesicles have an important role in cell metabolism and cell-to-cell communication. moreover, sevs when secreted from cells are capable to act as mediators of various neurological diseases. however, sevs show heterogeneity and this may impact their functions. therefore, to characterize sevs at a single-particle level is important to better detail the associated biological activity. in this scenario, we innovatively propose the structured illumination microscopy (sim) as a technique able to complement the non-optical methods (transmission electron microscopy, tem) to analyse single sevs and their markers. methods: human plasma sevs were separated from healthy cognitive control (ctrl), mild cognitive impairment (mci) and demented subjects. the sevscontaining pellet was resuspended in 4% paraformaldehyde or 2% glutaraldehyde solutions. for sim, sevsenriched preparations were washed with the blocking solution and incubated with the primary antibody (l1cam). the secondary fluolabelled antibody was then added. between the steps with the blocking solution, the primary and secondary antibodies, two ultracentrifuge steps were performed. the image acquisition was done on a nikon sim system with a 100x oil immersion objective. sevs were imaged with a 3d-sim acquisition protocol. tem was performed on 300 mesh formvar copper-coated grids. sevs-enriched preparations were incubated first with the blocking solution and then, immunogold-labelled for cd63. samples were counterstained with uranyl acetate and observed under a jeol 1010 ex electron microscope. data were recorded with a morada digital camera system. participation to the present study was approved by relevant local ethics committee of mendrisio and lugano hospital and written informed consents were obtained from subjects. results: for sim methodology, only vesicles in the range from 180 to 190 nm were detected, as the final resolution achieved was 160 nm. instead, for tem, sevs under 100 nm were identified. none of these methods provided relevant information about possible difference in morphology of the ctrl-, mci or demented subjects-derived sevs. summary/conclusion: even if both methods identified cd63 or l1cam-positive vesicles, sim resolution and the complexity of the protocol represent some disadvantages respect to tem, that may be the first choice screening technique for evs analysis to be then completed by sim for particular tasks. fabrication of nanopore structures via conformal metal-film deposition for ev sensing kwanjung kim a , seung-min han b , soo-hyun kim c and sung-wook nam d luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. these evsreleased under normal physiological conditions as well as in the pathogenesis of neurological, vascular, haematological, and autoimmune diseaseshave been shown to transfer biological molecules such as protein and rna between cells, potentially transmitting signals. to understand more about these signalling mechanisms, there is a need for detecting and quantifying evs with cargo protein and rna in a reproducible and reliable manner. however, this has been challenging due to the small size of evs (ranging from 30 to 100 nm in diameter), and the lack of specific staining reagents. methods: here, we utilize the amnis® cellstream® flow cytometer, which enables high-throughput flow cytometry with increased sensitivity for detecting small particles. we demonstrate that a charge-coupled device (ccd)-based, time-delay-integration image capturing system can be used to detect and quantify evs and their cargo labelled with exoglow™-protein or exoglow™-rna. results: in this study, we show flow cytometry data quantifying ev samples that have been labelled with cargo markers for proteins and rna. the ev cargo contents along with the appropriate control samples will be shown. summary/conclusion: the ccd based detection of the cellstream flow cytometer has the sensitivity to quantify evs and their cargo. single ev imaging reveals novel ev biomarkers and dna cargo siobhan m. king a , ricardo bastos a and andras miklosi b a oni, oxford, uk; b oni (oxford nanoimaging ltd), oxford, uk introduction: extracellular vesicles (evs) are cellderived membrane-bound particles that range in size from 30-1000 nm and carry active molecules such as dna, rna and proteins. upon secretion, evs can execute many biological functions such as initiating intracellular communication or regulating immune responses. depending on their origin evs have different characteristics and cargo, making them attractive candidates for early diagnostic and therapeutic applications. however due to their small size and heterogeneity, direct visualization and characterization of the surface markers expressed remains a challenge since these vesicles are below the resolution limit of standard light microscopy. methods: here, we describe a method that provides size analysis of single-evs, which falls below the diffraction limit of light. this was done with purified evs, immunostained using fluorescently labelled primary antibodies raised against ev surface markers (cd63, cd81, cd9), specific cargo such as dna and probed for tissue specific cargo. characterization of the molecular content and structural properties of surfaceimmobilized evs was performed using single-molecule localisation microscopy (smlm) on the nanoimager platform. results: multicolour smlm was used to detect up to three ev biomarkers showing successful characterization of the molecular signature for different ev subpopulations. the distribution of novel components on urinary evs were visualized for the first time using this approach. in addition, smlm revealed the presence of dna on both the surface and also as a cargo inside evs isolated from tumour cell culture media, which was validated using complementary biochemical characterization. summary/conclusion: smlm is a powerful technique for single-ev analysis and characterization. visualization of single-urinary evs enabled accurate sizing and further insights into novel components expressed on the subpopulation's membrane surface. together, the data demonstrates that the quantitative abilities of smlm can significantly enhance our understanding of evs, as structure, phenotypes, and cargoes can now be successfully resolved. introduction: working skeletal muscle is a common site for injury due to unaccustomed exercise with or without underlying pathology. direct analysis of skm injury requires invasive tissue biopsies. circulating extracellular vesicles (evs) are abundant in blood and have been shown to be enriched in microrna; profiles of which may reflect the state of tissues. evs may therefore serve as a non-invasive indicator of muscle injury and regenerative processes in vivo. methods: two consecutive bouts of muscle-damaging exercise (plyometric jumping and downhill running) were performed by 9 healthy male volunteers. serum creatine kinase (ck) and plasma evs were analysed at baseline, 2 and 24 hr post-exercise. perceived muscle pain (pmp) was assessed at 2 and 24 hr post-exercise. large evs were isolated using a 10 000 g centrifugation step, and small evs were isolated using qev columns. ev-enriched isolates were visualized using tem, and size and numbers were quantified using nta. based on nta results the highest particle fractions (7-10) were pooled for rna analysis. qpcr was done on plasma, large evs and small evs. a group of muscle and immune cell-important mirs were analysed by means of normalization to an exogenous control. results: ck and pmp increased post-exercise, providing evidence for muscle damage. tem revealed an abundant and heterogeneous pool of evs. a concomitant abundance of evs was seen with nta (mean = 1.17 x 1010 particles/ml plasma). mean ev diameters were 194 ± 22 nm across all timepoints. no change in ev size nor number was seen over time, however, mir-31 decreased at 24 hr when compared to 2 hr in the small ev isolate only. plasma displayed an immediate increase in myomirs-1 and −206 at 2 hr, which returned to baseline at 24 hr. in contrast, myomirs-208b and 486 remained elevated over the 24 hr period. myomir-133b and 486, as well as immune-mirs, did not change in evs or plasma as a result of the intervention. summary/conclusion: the decrease in mir-31 in small evs at 24 hr is consistent with previous data. no decrease in mir-31 in large evs suggests specific packaging and hence a specific response to the muscle damage in small evs. more changes occurred in plasma myomirs suggesting less specific passive leakage into circulation from damaged cell membranes. funding: south african national research foundation pulsed electromagnetic fields potentiate the paracrine function of mesenchymal stem cells for cartilage regeneration yingnan wu a , dinesh parate a , eng hin lee a , zheng yang a and alfredo franco-obregón b a national university of singpaore tissue engineering program, yll school of medicine., national university of singapore, singapore, singapore; b biolonic currents electromagnetic pulsing systems laboratory, biceps, national 11 university of singapore, singapore, singapore introduction: the mesenchymal stem cell (msc) secretome, via the combined actions of its plethora of biologically active factors, is capable of orchestrating the regenerative responses of numerous tissues by both eliciting and amplifying biological responses within recipient cells. mscs are "environmentally-responsive" to local microenvironmental cues and biophysical perturbations, influencing their differentiation as well as secretion of bioactive factors. we have previously shown that exposures of mscs to pulsed electromagnetic fields (pemfs) enhanced msc chondrogenesis. here, we investigate the influence of pemf exposure over the paracrine activity of mscs and its significance to cartilage regeneration. also, the subsequent extracellular vesicles analysis and isolation are processed for the understanding of how the pemfs affect stem cell evs and consequent differentiation induction. methods: conditioned medium (cm) was generated from mscs subjected to either 3d or 2d culturing platforms, with or without pemf exposure. the paracrine effects of cm over chondrocytes and msc chondrogenesis, migration and proliferation, as well as the inflammatory status and induced apoptosis in chondrocytes and mscs was assessed. the cms which have significant effects during chondrogenesis will be analysed by protein and mirna studies. results: we show that the benefits of magnetic field stimulation over msc-derived chondrogenesis can be partly ascribed to its ability to modulate the msc secretome. mscs cultured on either 2d or 3d platforms displayed distinct magnetic sensitivities, whereby mscs grown in 2d or 3d platforms responded most favourably to pemf exposure at 2 mt and 3 mt amplitudes, respectively. ten minutes of pemf exposure was sufficient to substantially augment the chondrogenic potential of msc-derived cm generated from either platform. furthermore, pemf-induced cm was capable of enhancing the migration of chondrocytes and mscs as well as mitigating cellular inflammation and apoptosis. the cms protein results in the significant promotion chondrogenesis condition showed an increase in proliferation and anti-inflammatory cytokines. summary/conclusion: the findings reported here demonstrate that pemf-stimulation is capable of modulating the paracrine function of mscs for the enhancement and re-establishment of cartilage regeneration in states of cellular stress. the pemf-induced modulation of the msc-derived paracrine function for directed biological responses 1 in recipient cells or tissues has broad clinical and practical ramifications with high translational value across numerous clinical application. effects of extracellular vesicles from blood derivatives on osteoarthritic chondrocytes within an inflammation model introduction: the degenerative disease osteoarthritis (oa) is one of the leading causes of disability especially of elderly people. besides various treatment options depending on the severity of the cartilage degradation, the application of blood derived products such as platelet rich plasma (prp) are getting more and more popular in clinical practice due to its high concentration of platelets and the perceived high growth factor levels. drawbacks of using prp include high donor variability, discrepancies among preparation protocols and the presence of cells (platelets, leukocytes) which can evoke cellular processes, especially inflammation, when injected into the diseased tissue. one possibility is to isolate only extracellular vesicles (evs) from blood derivatives to overcome these problems. in the current study the effects of evs isolated from blood derivatives on oa chondrocytes within an inflammation model was investigated. methods: cd14 positive primary monocytes were isolated from citrate anticoagulated whole blood by magnetic bead sorting. monocytes were differentiated into resting m0 macrophages and activated into m1 macrophages according to published protocols. elisa measurements verified successful differentiation and activation as il1β and tnfα levels increased. as control, thp1 monocytes were used. patient-derived oa chondrocytes were grown in 6 well plates and co-cultivated with activated m1 macrophages which were seeded into thincerts and added to the 6 wells representing the inflammation model. furthermore, cells were treated for 48 hours with media containing fcs, ev depleted fcs or evs isolated from prp or hypact serum. results: successful differentiation and activation of monocytes (thp1 and primary monocytes) into m1 macrophages was demonstrated by elevated levels of the inflammatory cytokines il1β and tnfα. within the inflammation model (co-culture of oa chondrocytes with m1 macrophages), addition of evs isolated from prp or hypact serum resulted in decreased secretion levels of il1β and tnfα compared to media supplemented with either fcs or ev depleted fcs. summary/conclusion: taken together, evs from blood derived products might be chondroprotective and anti-inflammatory mediators which protect cartilage from being degraded during oa. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst3-f-5030664/003-2017. 1α,25(oh)2d3 regulates growth cartilage matrix vesicle micrornas niels asmussen, michael mcclure, zhao lin, zvi schwartz and barbara boyan virginia commonwealth university, richmond, usa introduction: matrix vesicles (mvs) are small (50-150 nm in diameter) lipid bound extracellular organelles isolated from calcifying tissues including the growth zone (gc) of growth plate cartilage. 1α,25 (oh)2 vitamin d3 (1α,25) is a regulator of gc chondrocytes and the mvs they produce. these mvs are key players in the mineralization process and are selectively enriched with enzymes and growth factors. we found that mvs are also selectively enriched with micrornas (mir), including mir-22, mir-122 and mir-451. the aim of this study was to determine the regulatory role of 1α,25 in the packaging of mirna in mvs by gc cells. methods: gc cells were isolated by enzymatic digestion from costochondral gc cartilage harvested from 5 wk-old male sprague dawley rats (iacuc approved). confluent fourth passage gc cell cultures were treated with 10-8 m 1α,25 or vehicle for 24 h. media were removed, cell monolayers digested with trypsin and cells and mvs isolated by differential ultracentrifugation. rna was precipitated from cells and mvs. small rnaseq data were trimmed, aligned and counted before undergoing differential expression analysis. experimental groups had an n = 3 per variable. significant differences (p < 0.05) were determined using r v 3.6.2. results: 1α,25 treatment altered expression of 30 mv mirs compared to control mvs, whereas 5 cell mirnas were differentially expressed. 62.1% of significantly up or down regulated mir found in mvs overlapped between 1α,25 and vehicle groups with the remaining being uniquely differentially expressed. 1α,25 increased mv mir-150 and decreased mir-384-3p two mirs known to regulate osteoblast proliferation (150 increases, 384 decreases). summary/conclusion: 1α,25 regulates gc chondrocyte and mv behaviour and this study demonstrates that it also impacts the mir packaging within mvs. mir discovered in mvs have been demonstrated to impact chondrocyte behaviour and the present study indicates that 1α,25 regulates the growth plate through mir delivered by mvs. introduction: increasing evidence has proposed extracellular vesicles (evs) as mediators of many of the therapeutic features of mesenchymal stromal cells (msc) that have been widely studied in clinical trials over the last years. these evs have been recognized as nanocarriers of important biological information, which play a central role in cell-to-cell communication. in this context, evs can be used as an alternative to a cell-based therapy, with reduced risks. the present work aimed to evaluate the impact of different culture conditions on the msc-derived evs molecular composition through fourier-transform infrared (ftir) spectroscopy. methods: evs derived from msc from different sources, expanded in two different culture media ((xenogeneic -free (xf) vs serum-containing medium (fbs)) were characterized by ftir spectroscopy, a highly sensitive, fast and high throughput technique. moreover, principal component analysis (pca) of preprocessed ftir spectra of purified evs was conducted, enabling the evaluation of the replica variance of the evs chemical fingerprint in a reduced dimensionality space. for that, different pre-processing methods were studied as baseline correction, standard normal variation and first and second derivative. results: evs secreted by mscs cultured with serumcontaining medium presented a more homogenous chemical fingerprint than evs obtained with xf medium. the regression vector of the pca enabled to identified relevant spectral bands that enabled the separation of samples in the score-plot of the previous analysis. ratios between these spectral bands were determined, since these attenuate artefacts due to cell quantity and baseline distortions underneath each band. statistically inference analysis of the ratios of spectral bands were conducted, by comparing the equality of the means of the populations using appropriate hypothesis tests and considering the significance level of 5%. it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture medium used for msc expansion and the msc donor. summary/conclusion: this work is a step forward into understanding how different culture conditions affect msc-derived evs characteristics. funding: fundação para a ciência e tecnologia (ptdc/equ-equ/31651/2017, uidb/04565/2020). performance qualification for microflow cytometers: understanding technical limitations to improve your research desmond pink a , michael wong a , diana pham a , renjith pillai a , leanne stifanyk b , sylvia koch b , rebecca hiebert a , oliver kenyon c and john lewis a a nanostics, edmonton, canada; b dynalife, edmonton, canada; c apogeeflow systems, hemel hempstead, uk introduction: as microflow cytometry and other techniques mature as validated modalities for analysing extracellular vesicles (ev), there has been a concerted effort to improve reproducibility . in order for this reproducibility to occur there has to be a critical understanding of advantages and limitations for each technology. for microflow cytometry, several instruments are available to analyse evs. each platform has different limitations as well as advantages over other platforms. to provide the optimal data for your specific research, it is critical to understand the limitations of your platform. to accurately define these limitations, a performance qualification (pq) of your instrument should be undertaken. methods: an apogee a60 platform was used in these experiments. experiments were designed with expected ranges and cut-offs for acceptance criteria.initial tests included autosampling of a 96 well plate with either single or double aspiration, single sample reproducibility and linearity proportional to flow rate. other experiments designed to show machine performance included minimal time to achieve valid data, sample volume required for double aspiration, determination of coincidence; detection sensitivity using a spiked sample; flow rate stability for extended periods (5-30 minutes) . tests should also be performed to determine carryover at a range of sample concentrations. if present, the means to remove contaminating samples should be determined. any performance tests should be applicable to any instrument in the field. results: auto-sampling helped demonstrate consistent data; reproducibility of total events and biomarkers was 2-8% c.v. detected bead concentrations were linear with flow rates between 0.75 and 10.5 ul/min. double well aspirations provided similar data with aspirations between 60-80ul. valid data was achieved for a low abundant target (~200-400 events/ul) after only 30 s, <10%c.v. detection sensitivity was determined to be~1/400,000. carryover ranges were determined in the presence of nominal unstained serum. an optimal number of machine washes was determined. some membrane stains, such as cell mask and cfse require much more rigorous cleaning to remove stain carryover. summary/conclusion: to improve data reproducibility, performance qualification of any instrument is key. operational limitations help define optimal performance parameters of any technology. understanding the types of experiments to perform for your particular type of characterization technology depends on the requirements you set for your research. a good performance test should be applicable to any related instrument in the field. funding: funding provided by nanostics, the alberta cancer foundation, and alberta innovates. introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par20-053, is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par16-267/ 277, successfully funded 7 r01 and 4 r21 grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, 2018); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, 2019). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, 2019). summary/conclusion: drs. sudhir srivastava and matthew young are the programme directors for the par which began accepting applications on 5 january 2020. this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute. introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of 7 protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores (600 nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the 7 protein biomarkers (tau, uchl-1, nfl, gfap, il6, il10, and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl1 were elevated in mtbi patients compared to controls (p < 0.05), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il6 and tnf-with tau from glur2+ evs showed 88% accuracy with 80% sensitivity and 100% specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. ps15.05 = op3.05 l1cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma maia norman, dmitry ter-ovanesyan, wendy trieu and david walt wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l1cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l1cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l1cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l1cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd9, cd63 and cd81) as well as l1cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l1cam appears. we also immunocapture l1cam from csf and plasma and perform western blots for the internal and external domains of l1cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l1cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l1cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l1cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l1cam in plasma is likely not coming from the brain. this data call into question the utility of l1cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant 3d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed 3d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in 6-month-old wt mice, (n = 6/groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for 15% of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and nonamplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed 968 proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy5y cells with n-myc amplified sk-n-be2 cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be2 cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. ps15.08 = op3.08 results: murine ctl evs were broadly divided into two populations that were eluted at low salt (l-s: 0.15 m-0.3 m nacl) and high salt (h-s: 0.3 m-0.5 m nacl) concentrations. l-s ctl evs were abundant in late endosome-related proteins, integrins, rabs, and effective mirnas, indicating exosome characteristics, and had biological activity for preventing tumour metastasis after depletion of tumoural mesenchymal cell populations by intratumoral administration (see seo et al., nat. commun. 9: 435, 2018) . contrary, h-s ctl evs were rich in dna, core histones, ribosomal proteins, cytoskeleton proteins, and housekeeping proteins, considering microvesicles and apoptotic bodies, and easily phagocytosed by a kupffer cell line (kup5: kitani et al., results immunol. 4: 68-74. 2014 ). in addition, there were noticeable differences between ls and h-s ctl evs in the negative zeta potential width and membrane glycan structure. summary/conclusion: thus, ion exchange can be an optimal mass fractionation method for discriminating bioactive exosomes from cargos for nucleic acids in evs. funding: cryotem was conducted in nara institute of science and technology (naist), supported by nanotechnology platform program (synthesis of molecules and materials: 2019 #04) of the ministry of education, culture, sports, science and technology (mext). this work was supported by grants from the japan agency for medical research and development (translational research network program (nagoya univ. seeds a64)) and the japan science and technology agency (crest [jpmjcr17h2]). clic4 is essential for breast cancer metastatic competence and predicts disease outcome introduction: metastatic breast cancer is a consequence of complex interactions between cancer cells and the host. clic4, a member of a conserved gene family in the glutathione-s-transferase superfamily, mediates crosstalk between tumour and host in breast cancer. tcga and metabric data indicated that elevated clic4 expression was associated with breast cancers from young women, those with poor prognosis, and those with early stage metastatic disease. methods: since bulk tumour analysis does not distinguish between cancer and host stromal cells, we used genetic modifications of established syngeneic breast cancer mouse models to evaluate the contributions of clic4 in the host or tumour cells to develop metastases. results: experimentally, the essential clic4 host contributions for metastatic competence were related to circulating levels of pro-metastatic soluble factors, neoangiogenesis, tumour cell attachment to lung tissue, myofibroblast differentiation, and leukocyte migration. clic4 was detected as cargo in circulating extracellular vesicles (evs) from breast cancer patients. similarly, circulating evs from tumour-bearing mice have abundant clic4 in comparison to those from mice bearing tumours that lack clic4. tumour cells released evs that induced myofibroblast conversion of wildtype but not clic4 ablated lung fibroblasts. summary/conclusion: these results illuminate clic4 expression as a prognostic marker for breast cancer patients, and experimentally, clic4 is a critical host factor for metastatic competence and potential target within host tissues for anti-metastatic therapy. funding: this work was supported by the intramural program of the national cancer institute under project zia bc 005445. the application of flow cytometry in an ev-based liquid biopsy for the detection of cancer multidrug resistance in myeloma gabriele de rubis a , krishna sunkara a , sabna rajeev krishnan and mary bebawy b a laboratory of cancer cell biology and therapeutics, discipline of pharmacy, graduate school of health, the university of technology sydney, australia, sydney, australia; b the university of technology sydney, sydney, australia introduction: multiple myeloma (mm) is an incurable cancer of bone-marrow plasma cells. it is characterized by unpredictable and highly variable therapeutic response and poor survival, attributed to the development of multidrug resistance (mdr) to chemotherapy. presently, no clinical procedures allow for a continuous, minimally invasive monitoring of mdr. we identified unique extracellular vesicle (ev) populations in the blood of myeloma patients, which serve as biomarkers of disease evolution and mdr to combination chemotherapy. we describe approaches used to optimise the use of flow cytometry (fcm) for ev summary/conclusion: although further investigation is required, our results potentially promise an effective and inexpensive priming agent (i.e., ethanol) for the production of anti-inflammatory msc-evs. this, combined with the significant increase in yield via 3d dynamic culture, presents practical solutions to both ev manufacturing scalability and potency issues. donor source affects potency of mesenchymal stem cell-derived extracellular vesicles introduction: mesenchymal stem cell (msc) therapies have been heavily investigated for their utility in applications such as wound healing and regenerative medicine due to their angiogenic, immunomodulatory and anti-apoptotic effects. recently, msc-derived extracellular vesicles (evs) have been implicated as primary effectors in msc-based therapies via protein and nucleic acid cargo transfer to patient cells. msc evs represent a superior alternative to msc-based therapies, as they lack the ability to replicate and are much smaller in size, circumventing related safety concerns such as immunogenicity, teratoma formation and blood vessel occlusion. however, a key drawback with msc therapies in general is their variable therapeutic potency, which is dependent on donor source. as a cell derived therapeutic, this crucial limitation is hypothesized to exist in msc evs as well. here, we demonstrate the varying bioactivities of isolated msc evs from differing donors and tissue sources. methods: six separate msc lines were obtained from different donors, with three msc lines derived from donor adipose tissue, and the other three from the bone marrow of separate individuals. evs were isolated from each msc line at passage 3 via differential centrifugation and ultrafiltration. these isolated msc evs were then characterized for size/concentration via nanoparticle tracking analysis, and ev markers (tsg101, alix, cd63) via western blot. pro-vascularization capacities of msc evs were determined by a gap closure assay using human umbilical cord vein endothelial cells (huvecs). results: characterization of msc evs revealed similar sizes and ev marker expression across donor groups, frontiers in chemistry, submitted funding: this work was funded by the momentum programme (lp2016-2), by the national competitiveness and excellence program catalan institution for research and advanced studies (icrea) proteomic profiling of retinoblastoma-derived exosomes reveals potential biomarkers of vitreous seeding angel montero carcaboso g , andrea petretto b , franco locatelli a and angela di giannatale a a department of paediatric haematology/oncology and cell and gene therapy, irccs, ospedale pediatrico bambino gesù ps08: separation and concentration a laboratory of clinical biophysics, faculty of health sciences ps10: diverse ev biomarkers chair: pia siljander -faculty of biological and environmental sciences urinary evs were isolated using low vacuum filtration method followed by ultracentrifugation. raman spectra of urinary evs were recorded using a renishaw invia raman spectrometer. data analysis was performed using principal component analysis (pca) and hierarchical cluster analysis (hca). the size distribution and morphology of evs were analysed by transmission electron microscopy and nanoparticle tracking analysis methods. results: average raman spectra obtained for urinary evs from studied groups showed differences in intensities of specific bands in the region of 400-1800 cm-1. we found significant correlations between mean area under curve (auc) calculated for raman bands (phenylalanine, dna, proteins, lipids and amide i) and selected clinical parameters such as: egfr, serum creatinine, glucose, urine creatinine. chemometric methods showed spectral pattern responsible for separation between studied groups. nta measurements visualized evs with size of 204.7 ± 96.5 nm. summary/conclusion: our results showed that characteristic raman spectra of urinary evs are promising candidates for new, non-invasive biomarkers for dkd isolation of circulating extracellular vesicles and cfdna allows for erbb2 detection in a single aliquot of breast cancer patients plasma michela notarangelo a , mattia barbareschi b , antonella ferro c , orazio caffo c , vito d'agostino a and francesca demichelis d a department of cellular results: results: tissue-derived large and small evs showed difference in size (mean 142 nm vs 75 nm) when examined by em, whereas nta and exoview™ were unable to show a clear difference between the populations (nta: mean 125.7 nm vs 122 nm nta can only detected vesicles above 70 nm and exoview™ only measures vesicles between 50-200 nm. of the three different methods, em analysis of single vesicles visualized in a significant number of micrographs was the only one able to distinguish ev subpopulations by size. funding: funding: swedish research council knut och alice wallenberg foundation imaging of human plasma-derived small-extracellular vesicles using transmission electron microscopy and structured illumination microscopy mitovesicles: a new extracellular vesicle of mitochondrial origin altered in ageing and neurodegeneration alldred b , chris goulbourne b , hediye erdjument-bromage d , monika pawlik b , mitsuo saito e , mariko saito f , stephen d. ginsberg b an in vitro and in vivo perspective on the role of erythrocyte-derived extracellular vesicles in parkinson's disease pathology frédéric calon c , éric boilard f and francesca cicchetti b a centre de recherche du chu de québec and faculté de médecine, département de psychiatrie & neurosciences département de microbiologie-infectiologie et d'immunologie evidences on microalgal extracellular vesicles: a morphological assessment antonella bongiovanni i , ales iglič j and veronika kralj-iglič j a laboratory of clinical biophysics, faculty of health sciences a faculty of dentistry, national university of singapore, singapore, singapore, singapore; b institute of medical biology, agency for science, technology and research, singapore, singapore, singapore; c exosome of cancer-associated fibroblast induce anti-cancer drug-resistance of nsclc so-young kim a and yeon-ju lee b a chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea; b chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea introduction: the understanding of interaction mechanisms between cancer cells and the tumour microenvironment (tme) is crucial for developing therapies that can arrest tumour progression and metastasis. cafs are the major constituent of the tme in many cancers. recent studies indicate that exosomes harbour the potential to regulate proliferation, survival and immune status in recipient cells. most of the current studies are focused on cancer cell secreted exosomes; and little is known about cafderived exosomes and their influence on cancer cells. methods: nsclc cell lines (pc9gr) and mrc5 (normal fibroblast cells) were grown in culture with exosome-free fbs. cutured media was filtrated by tangential flow filtration systems. exosomes in supernatant were isolated with the exoquick-tc™ system. considering the important role of cell extrinsic factors on cell growth and survival, we assessed whether factors contained in the mpa exosome could affect proliferation and survival of recipient cancer cells. cells were then treated with 1 μm osimertinib or pbs for 3 days prior to cell quantification of live cells.to investigate mechanisms of resistance to osimertinib mediated by ma or mpa-exosome in nsclc cell lines, we test cell viability by crystal violet assay in trametinib or osimertinib treated after pretreated ma or mpa-exosome, pc9gr during 6 days. we will investigate how mpa-exsomes activate erk signalling pathway in pc9gr cells to induce antitumor effects by western blot. results: mpa exosome increased proliferation of pc9gr cells by more than 50% compared to control pbs. pc9gr cells grown in mpa-exosome and subsequently treated with osimertinib showed a significant increase in cell survival compared to pc9gr cells grown in ma-exosome. osimertinib is used to treat egfr-mutant non-small cell lung cancer (nsclc) with tyrosine kinase inhibitor resistance mediated by the egfr t790 m mutation.these data show that "mrc5-pc9gr-crosstalk factors" affect proliferation and adaptive drug resistance of cancer cells. mpa-exosome mediates erk signalling activation and attenuated after treatment of 1um osimertinib. summary/conclusion: cafs support cancer growth and invasion. co-cultured nsclc with mrc5 lung fibroblast increased cell viability and exosomal mir-21 through the tgf-ß pathway in treatment osimertinib. exosomal mir-21 up-regulation in cocultured nsclc with mrc-5 induced drug resistance to drug-induced apoptosis. thus, exosomal mir-21 expression in co-cultured nsclc with mrc5 may support drug tolerance persister cells. introduction: neural stem cell (nsc) therapy has shown promise for brain repair after injury or disease mostly through bystander effects. nevertheless, the translation of nscs derived from human induced pluripotent stem cells (hipscs) to the clinic remain constrained due to safety issues, which include immunogenic risks, tumorigenesis potential, and incomplete differentiation. a way to avoid these issues is by using extracellular vesicles (evs) generated from nscs, as nsc-evs likely have similar neuroprotective properties as nscs and are amenable for non-invasive administration as an autologous or allogeneic off-the-shelf product. however, this would require reliable purification and characterization processes, and testing of evs for composition and biological properties. methods: we generated evs from hipsc-derived nscs using a combination of ion-exchange chromatography (iex) and size-exclusion chromatography (sec) and investigated their composition through small rna sequencing and proteomics. we also performed in vitro and in vivo experiments to determine their biological and functional properties. results: iex and sec facilitated purification of hipsc-nsc evs nearly to homogeneity, which expressed ev markers such as cd9, cd63, cd81, and alix with a mean size of 145 nm. small rna sequencing revealed enrichment of mirnas related to different neuroprotective signalling pathways and diverse metabolic functions consistent with their role in cell-cell communication. the proteomic analysis identified >1,000 proteins, including ev markers and many other proteins involved in central nervous system function and cellular processes. the evs also displayed antiinflammatory activity in an in vitro mouse macrophage assay. intranasal (in) administration of nsc-evs resulted in their rapid incorporation by neurons, microglia, and astrocytes in virtually all regions of the brain. functionally, in administration of nsc-evs reduced inflammatory activity in the brain in a model of status epilepticus, and increased hippocampal neurogenesis in the adult brain. summary/conclusion: biologically active evs with antiinflammatory and neurogenic properties could be purified and harvested from hipsc-nscs. such evs also contain many mirnas and proteins that are of interest for brain repair after injury or disease. funding: supported by a grant from the national institute of neurological disorders and stroke (1r01ns106907-01 to a.k.s.) introduction: extracellular vesicles (evs) generated from human bone marrow-derived mesenchymal stem cells (hmscs) display anti-inflammatory and neuroprotective properties. our recent study has shown that intranasally (in) administered hmsc-evs incorporate into significant percentages of neurons and microglia in virtually all regions of the intact as well as the injured forebrain within 6 hours (kodali et al., int j mol sci, 2019) . in this study, using a rat model, we investigated the efficacy of a low dose of hmsc-evs administered intranasally for alleviating the abnormal plasticity of newly born neurons and the activation of microglia after se. methods: approximately 10 billion evs were dispensed bilaterally into both nostrils of young f344 rats that experienced two hours of kainate-induced se. animals were euthanized seven days after se, and brain tissue sections were processed for immunohistochemical staining of neun (a neuronal marker), dcx (a marker of newly born neurons), iba-1 (a microglial marker), and parvalbumin (pv) and reelin (markers of subclasses of interneurons). in addition, activated microglia were quantified using iba-1 and cd68 dual immunofluorescence. results: in administration of evs reduced the seinduced loss of pyramidal neurons in the hippocampal ca1 subfield. also, ev administration after se maintained higher levels of pv+ interneurons in the dentate gyrus. furthermore, ev treatment after se modulated abnormal neurogenesis, which was evidenced by a the role of small extracellular vesicles in chronic neuropathic pain zhucheng lin a , renee jean-toussaint b , yuzhen tian b , ahmet sacan a and seena ajit b a drexel university, philadelphia, usa; b drexel university college of medicine, philadelphia, usa introduction: chronic pain is the most prevalent, disabling, and expensive public health condition in the usa. exosomes are 30-150 nm extracellular vesicles that can transport rnas, proteins, and lipid mediators to recipient cells via circulation. exosomes can be beneficial or harmful depending on their source and contents. we hypothesized that the composition of small extracellular vesicles (sevs) can be altered following nerve injury and these alterations can provide insight into how the body responds to neuropathic pain. methods: to characterize changes following nerve injury, small extracellular vesicles (sevs) were purified by ultracentrifugation from mouse serum four weeks after spared nerve injury (sni) or sham surgery. mirna profiling and proteomics analysis using tandem mass spectrometry were performed to determine differential expression of mirnas and protein cargo respectively. for in vivo studies, sevs were administrated intrathecally into the mouse lumbar region. animals were evaluated for mechanical and thermal hypersensitivity over 21 days after injection. results: our mirna profiling showed a distinct mirna signature in sni model compared to sham control. proteomics analysis detected 274 gene products. of these, 24 were unique to sni model. neuropathic pain can induce the activation of the complement cascade and we observed significant upregulation of complement component 5a (c5a) in sevs from sni model. intercellular adhesion molecule 1 (icam-1), required for the leukocyte recruitment, adhesion and homing of exosomes was also upregulated in sevs from sni model compared to sham control. administration of sevs from sni model increased paw withdraw threshold in naïve recipient mice and inflammatory pain model, indicating a protective role for sevs in attenuating chronic pain. summary/conclusion: our preliminary studies suggest a critical role for sevs cargo in regulating pain. additional studies are ongoing to determine the functional significance of alterations in sevs composition using mouse models of pain. introduction: amyotrophic lateral sclerosis (als) is a progressive adult-onset neurodegenerative disease caused by selective motor neurons (mns) death. the rapid disease progression strongly suggests that cell-tocell spreading of noxious factors could take place in als pathogenesis. extracellular vesicles could potentially spread the disease. in this study, we characterized large (levs) and small extracellular vesicles (sevs) isolated from plasma of sporadic als patients and healthy controls and determined their different composition in order to understand their neuroprotective or neurotoxic role in als pathogenesis. methods: levs and sevs were isolated from plasma of 40 als patients and 30 healthy volunteers by differential centrifugation and characterized by nanosight ns300. cd45, cd31, cd61, cd235a and annexin v were used for flow cytometry. sod1, tdp43, fus protein level was investigated by western blot. for raman spectroscopy, evs were dried on top of a caf2 slide and raman spectra were acquired using a 633 nm laser line. mirna libraries were prepared by truseq small rna library kit (illumina). results: the mean size both for levs and for sevs resulted increased in als patients compared to controls. levs derived from als patients were enriched in sod-1, tdp-43 and fus proteins compared to ctrls. sevs showed a distinct spectral pattern from levs. in addition, levs of als patients were richer in lipids and had less intense bands relative to aromatic aminoacids compared to healthy controls. we also found a great presence of leukocyte derived levs (lmvs) in als patients compared to ad patients and healthy donors and significant correlation with the progression rate of the disease. on the other hand, mirna and rna whole transcriptome sequencing identified a specific signature of mirnas in plasma derived sevs of als patients compared to a group of healthy controls and three neurological groups of control. summary/conclusion: these data may suggest that levs derived from als patients, enriched in lipids and toxic proteins, might play a role in prion-like propagation and immunity of als disease, while sevs, deriving ps05.07 introduction: dendritic spines are actin-rich structures at the postsynaptic sites of most excitatory synapses in the central nervous system. they are highly important structures for higher brain functions such as learning and memory. several live imaging studies have shown that long, thin, actinrich protrusions called dendritic filopodia are precursors of dendritic spines in hippocampal and cortical neurons. so far, many intracellular factors that regulate filopodia formation have been identified. however, extracellular mechanisms of filopodia formation are largely unknown. also, detailed molecular mechanisms by which astrocyte secreted factors regulate synaptogenesis are not well understood. small extracellular vesicles (sevs)/exosomes have potential to regulate filopodia, spine and synapse formation in autocrine or paracrine manner due to their unique cargo composition. here, we examine role of exosomes in filopodia, spine and synapse formation. methods: primary rat hippocampal and cortical neurons were transiently transfected with the multivesicular body (mvb) docking regulator gfp-rab27b or with shrnas against the exosome secretion and biogenesis regulators rab27b and hrs. transfected neurons were immunostained for synaptic proteins and analysed for filopodia at day in vitro (div) 6 or spines at div12. for rescue experiments, exosomes were isolated using differential ultracentrifugation method from conditioned media of div9 cortical neurons or primary astrocytes and characterized for their size, common protein markers and morphology. results: here, we find that mvb docking factor gfp-rab27b localizes to both the tips and bases of actin-rich filopodia and spines in primary neurons. furthermore, genetic regulation of exosome secretion by overexpression or knockdown of rab27b or hrs leads to respective increases or decreases in the number of filopodia, spines and synapses. the defects of exosome-inhibited neurons in filopodia density are rescued by add-back of neuronal exosomes. additionally, treatment of primary neurons with exosomes isolated from primary astrocyte cultures leads to enhanced spine and synapse formation. summary/conclusion: these results indicate that autocrine and paracrine communication via exosomes are a key part of the process of neuronal filopodia, spine and synapse formation. effects of apolipoprotein e genotype on protein and small rna profiles of brain tissue-derived extracellular vesicles of alzheimer's disease patients introduction: multiple sclerosis (ms) is the most frequent chronic inflammatory disease of the young adult central nervous system. nevertheless, the pathogenesis remains largely unknown. it is therefore relevant to better characterise in cerebrospinal fluid (csf), which irrigates the brain, novel bioactive compounds whose dysregulation could be involved in ms pathology. the concentration of extracellular vesicles (evs) has been already found affected in ms patient fluids but the content in bioactive molecules, particularly the micrornas (mirnas), remains barely investigated. the mirna are short oligonucleotides that are major posttranscriptional regulators and we previously showed the dysregulation of specific mirnas in csf of ms patients. evs can potentiate mirna effects by allowing remote action through the shuttling within biological fluids such as csf while providing a protection from circulating rnase. nevertheless, csf remains a challenging fluid to analyse due to limited access, low volume and presence of lipoproteins (other putative mirna carrier) that can be co-isolated with evs. methods: we performed a comparative analysis of ev isolation from csf by size-exclusion chromatography (sec), density-gradient ultracentrifugation, ultrafiltration or chemical precipitation (chemp) to determine the optimal technique(s) to enrich ev. results: sec applied on csf of control patients showed optimal ev purification with sufficient evs from 0.5 ml of csf for downstream ev characterization. furthermore, we were able to isolate mirnas from csf and determined their enrichment in evs by rnase-sensitivity treatments. finally, we have combined chemp and sec to enable a fast and largescale isolation of evs from > 5 ml of csf, which successfully provided an increase in particles detected by nanoparticle tracking analysis. we are currently characterising the particles to confirm that they are purified evs, cleared from contaminants. summary/conclusion: this work opens perspective to analyse evs from ms patients and to determine whether mirnas participates in ms pathogenesis through their transit in evs. funding: fondation louvain, charcot foundation. differences in circulating number of extracellular vesicles between contact sport athletes with and without acute mtbi: a pilot study meghan rath a , jacqueline sayoc a , soo-young choi a , karlee burns b , aja corchado c , jane mcdevitt b , jingwie wu d , ryan tierney b , michael selzer e , xiaoxuan fan f and joon-young park a for bottom-up guc, increasing iodixanol gradients with 2.3 ml of samples were centrifuged at 186 k g for 18 h. fractions were then pooled based on densities (1.1-1.2 g/ml). bca and sds-page were used to analyse total protein; nanoparticle tracking (nta) and transmission electron microscopy (tem) for ev presence; and immunoblotting and imaging flow cytometry (ifcm) to evaluate ev specific markers. (ev-track id: ev190096). results: immunoblotting showed absence of actinin4 from all samples, while cd63 and tsg101 were detected for all samples; apart from imf_ip. nt_samples were not analysed reliably by nta and ifcm, due to the high concentration of casein micelles present (~10^14/ml in milk) that otherwise would be co-counted with evs. as expected, following ip, which most efficiently removed casein micelles, bca showed that samples had lowest total protein. this was confirmed by sds-page. thus, most effects were then focused on the ip casein-depleted samples. ifcm indicated that, post-guc, sm_ip evs had significantly (p < 0.05) more cd81-positive particles/ml of milk vs all other guc and 200 kduc samples. while there were no significant differences in sizes of ev separated from sm or imf, directly comparing the ip pre-treated samples, sm had significantly (p < 0.01) higher quantities of evs when compared to imf. additionally, tem indicated that evs separated from sm by guc were intact with limited background debris, whereas those separated from sm by duc and all imf evs were not. summary/conclusion: in conclusion, regardless of the method used, imf has fewer intact evs compared to sm. also, to obtain purest sm evs, ip followed by guc separation is optimal. introduction: extracellular vesicles (evs) exist as subpopulations with heterogeneous content. the surface heterogeneity of evs may reflect differences in functionality between ev subpopulations, as interactions with recipient cells may differ between ev subpopulations with different surface profiles. however, it is currently challenging to study functional differences between ev subpopulations due to the lack of suitable techniques to purify intact evs based on their surface signature. here, we showcase a novel capture-and-release platform to enrich intact ev subpopulations by their surface profile and compare their characteristics. methods: mda-mb-231 and skov-3 cell-derived evs were isolated using size exclusion chromatography. ev subpopulations were enriched based on surface markers cd9, cd63, cd81 or phosphatidylserine (ps) using a novel magnetic bead-based capture-and-release platform. obtained evs were characterized by transmission electron microscopy (tem), nanoparticle tracking analysis (nta) and western blotting. evs were fluorescently labelled using pkh67 and celltracker deep red (ctdr) and their uptake by recipient cells was examined using flow cytometry. results: western blot analysis showed that ev subpopulations enriched for the selected tetraspanins and ps were successfully isolated using a novel capture-andrelease platform. interestingly, evs isolated based on ps exposure (ps+) lacked most canonical ev markers. all ev subpopulations showed intact, cup-shaped morphology when analysed by tem, but contained less protein contaminants compared to the initial ev isolate. ps+ evs were slightly larger than other ev subpopulations when analysed by tem and nta. to test the capacity of ev subpopulations to interact with recipient cells, evs were labelled with pkh67 and ctdr prior to subpopulation fractionation. after fractionation, ps+ evs showed a significantly higher ctdr/pkh67 ratio than other ev subpopulations as determined by fluorescence spectroscopy, suggesting higher esterase activity of ps+ evs compared to other tested subpopulations. furthermore, mda-mb-231derived evs isolated based on cd9 and cd81 expression were taken up more efficiently by hmec-1 and mda-mb-231 cells than evs isolated based on presence of cd63 or ps. summary/conclusion: using a novel technology to isolate ev subpopulations based on their surface profile, we here show that composition and cellular uptake efficiency differs between ev subpopulations. theoretically, this technology is applicable to any surface marker of interest, allowing its use to further establish ev surface-functionality relationships and enrich evs with desirable characteristics for therapeutic purposes. funding: this work was supported by a veni grant (no. 17296) of the dutch research council (nwo). aml were harvested from tib49 cells cultured in evfree medium using serial ultracentrifugation. hspc (ksl; lin-sca1+ ckit+) clonogenicity and inflammatory responses were assessed using colony-forming unit (cfu) assay and real-time polymerase-chain reaction, respectively. ifn-alpha receptor 1 (ifnar1) expression and intracellular reactive oxygen species (ros) levels were assessed by flow cytometry. dna damage were assessed by quantifying nuclear γ-h2ax using immunofluorescent microscopy. results: similar to evs derived from aml patients, tib49 ev-aml elicited double-stranded breaks in hspcs, and actively suppressed hspc clonogenicity. transcriptional profiling revealed that exposure to ev-aml induced the upregulation of several inflammatory mediators in hspcs, including isg15, il-6, ifnα, ch25 h. inflammatory signalling triggered by ev-aml did not depend on ifnα signalling as evident from suppression of clonogenicity in ifnar1-null hspcs as well as the lack of evs-induced stat1 phosphorylation or ifnar1 downregulation. instead, we found increased levels of ros following ev-aml exposure. summary/conclusion: our findings support a model whereby ev-aml inflammatory signalling and oxidative stress lead to dna damage in hspcs. introduction: basic leucine zipper atf-like transcription factor 2 (batf2) is implicated in inflammatory response and anti-tumour effects. although the tumour suppressive function of batf2 has been reported, its extracellular role in maintaining a non-supportive cancer microenvironment has not been explored. methods: in this study, we established gbm orthotopic and subcutaneous tumour models in nude and balb/c mice and flow cytometry analysis determined the batf2 inhibitory effects of mdscs recruiting. we used transwell assay to determine batf2-positive evs (evs-batf2) inhibitory of the chemotaxis of myeloid-derived suppressor cells (mdscs) in vitro. in addition, exo-counter detection during the development of the gbm-batf2 model to demonstrate evs-batf2 crosstalk with distant tissues. amd3100 blocking in tumour model confirms that evs-batf2 dominated by the sdf-1a/cxcr4 signalling pathway. in addition, exo-counter detection of evs in 32 pairs of gliomas in different stages proposes plasma-evsbatf2 (plevs-batf2) as a prognostic marker. results: we found that tumour-derived evs-batf2 regulate crosstalk between glioma cells and tumour microenvironment by inhibiting mdscs recruitment. evs-batf2 can be detected in plasma and bone marrow of glioma-bearing mice, this provides direct evidence that glioma-derived evs can communicate with distant site by crossing blood-brain barrier. besides, evs-batf2 injection significantly reduced sdf-1α expression in the tumour tissues. after blocking sdf-1α signalling by amd3100, the inhibitory effects of batf2 overexpression on mdscs recruitment were rescued. evs-batf2 inhibit mdscs recruiting and secreting mmp2, mmp9, and vegfa which promote gbm progression. strikingly, exo-counter detection of evs in 32 pairs of gliomas in different stages reveals that the number of plevs-batf2 can distinguish stage iii-iv glioma from stage i-ii glioma and healthy donors. summary/conclusion: our results suggest that evs-batf2 may be an effective circulating biomarker associated with glioma progression. of note, we are the first to determine the regulatory role of evs-batf2 in regulating tumour microenvironment and propose plevs-batf2 as a prognostic marker predicting glioma progression and candidate target for gbm therapy. introduction: electrofluidics is an emerging technology of combining electronics and nanofluidics. one important device in electrofluidics is an ion transistor in which the ionic current through a nanopore is regulated by gate voltage bias. here, we suggest a fabrication method of nanopore by introducing focused ion beam (fib) and atomic layer deposition (ald) to sense extracellular vesicle (ev) via metal electrode structures. methods: we deposited 100 nm-thick silicon-nitrite layers on both sides of silicon wafer by low-pressure chemical vapour deposition (lpcvd). we fabricated rectangular patterns by photolithography followed by reactive ion etching (rie) on the backside of the wafer. anisotropic silicon etching by koh was performed. the front side of the chip was patterned by photolithography followed by ti/au deposition for the fabrication of electrode structures. we drilled 50~100 nm pores in the si3n4 membrane by fib. by the ald process, we deposited highly-conformal metal film, either platinum (pt) or ruthenium (ru) to shrink nanopores by a self limiting process. results: we expect that the ion current through the nanopore is efficiently controlled by the gate-surrounding structures. the nanopore ion transistor can be used to count the number of evs. summary/conclusion: we suggest a fabrication method of nanopore ion transistors by introducing focused ion beam (fib) and atomic layer deposition (ald). this device will be applicable for single ev sensing. introduction: extracellular vesicles (evs) are key players in cell-cell communication and increasing evidence has shown that evs function in cancer by promoting cancer cell motility and metastasis. analysing tumour-derived evs in biofluids is attractive because it would be a novel approach to a non-invasive liquid biopsy. unfortunately, evs are highly heterogeneous. they vary greatly in size, lipid composition, and cargo and are difficult to distinguish from other small particles in complex biofluids. we have developed a novel flow cytometry method to generate a distinct ev fingerprint to profile biological specimens. methods: evs from cell culture media (purified and unpurified) and biological fluids (plasma and urine) were detected by flow cytometry using features on individual evs produced by intrinsic (cd63-phluorin) and extrinsic (lipophilic dye, di-8-anepps, and antibodies) fluorescent labels. ev subpopulations were visualized with dimensional reduction (t-sne and umap) of 20-150 features that defined the vesicle size, shape, and fluorescent emission spectra associated with the fluorescent marker. unsupervised density based clustering (hdbscan) in conjunction with supervised machine learning (xgboost) was subsequently used to define subpopulations. we refer to this method as "ev fingerprinting". results: ev fingerprinting was successfully used to detect evs in complex biological specimens and trace their differential enrichment through conventional purification methods. evs were readily distinguished from protein complexes, lipoproteins and non-lipid particles. calibration with externally validated purified ev, as well as size, lipid, and fluorescence standards enabled ev fingerprinting as a rigorous and reproducible method for resolving heterogenous ev samples. ev fingerprinting applied to conditioned medium from tumour cells and biological fluids from cancer patients reveals unique ev profiles generated by cancer, further supporting the potential of ev fingerprinting as a liquid biopsy. summary/conclusion: our single-ev analysis approach characterizes whole ev populations in complex biological fluids without the need for purification, reducing time intensive purification protocols and subsequent sample loss, permitting efficient analysis of liquid biopsy samples. detection and quantification of extracellular vesicles with cargo protein and rna using the amnis® cellstream® flow cytometer introduction: the particle size distribution (psd) of extracellular vesicles (evs) is commonly measured by tunable resistive pulse sensing (trps) and nanoparticle tracking analysis (nta). both trps and nta have limitations that hamper the accurate measurement of the psd of evs, specifically in the size range from 50 to 100 nm. an alternative technique for measuring the psd of evs is micro-fluidic resistive pulse sensing (mrps). because a standard operating procedure (sop) for characterizing evs by mrps is absent, we aim to establish a reliable sop to ensure reproducible psd measurements of evs by mrps. methods: measurements (n = 3) of red-blood cell, prostate cancer cell line supernatant, and human urine and plasma evs were acquired in 10 × 10 s acquisitions. two microfluidic cartridges were used to study a dynamic range of 50-450 nm. samples were diluted into phosphate buffered saline with different concentrations of tween 20 or bsa. because the excess of particles affects the detection limit, serial dilutions were performed to find the optimal dilution for each sample. data were evaluated using data viewer software. results: the optimal dilution was determined for each sample by maximizing the particle rate and minimizing the measurement time while preserving a robust detection limit of 50 or 65 nm. moreover, we developed a procedure to optimize the peak filter settings of data viewer by fitting data to normal distributions and identifying threshold values for signal-to-noise ratio, symmetry, and transit time within 99% confidence. summary/conclusion: we recommend to use 0.1% w/ v bsa in dpbs as sample diluent, because tween 20 affects evs as confirmed by flow cytometry. by using orthogonal techniques and well-characterized biological test samples, we developed and validated a sop for ev detection by mrps, thereby making mrps a valuable tool for ev researchers. real-time measurements of extracellular vesicles binding kinetics achieved through interferometric imaging in a multiplexed microarray modality introduction: extracellular vesicles are very promising diagnostic biomarkers. as a matter of fact, the properties of these biological nanoparticles depend on the health conditions of each individual. however, experiments that involve evs phenotyping are time consuming, due to 12 h-or overnight incubations. in order to get accurate results, maximizing binding efficiency is a necessity; that normally involves ensuring the saturation of the capture reaction, which can result in an unnecessarily long incubation time. with the ability of labelfree kinetic binding measurements using interferometric reflectance sensing in a microfluidic chamber, we perform an optimization of the incubation time in different flow conditions, while demonstrating a new way of multiplexing for real-time evs specific capture and detection.methods: all the real-time binding measurements were performed with the interferometric reflectance imaging sensor (iris). iris chips were first coated with an organic polymer (mcp-2), which provides an active surface for probe immobilization. then, antibodies against cd9, cd81, cd63 markers were spotted at different densities in a microarray modality. the chips were then encapsulated with a glass window to form a microfluidic chamber that allows for imaging the sensor surface. samples of hek-derived extracellular vesicles were flowed across the sensor surface in the iris system and real-time images were acquired. incubation was performed at different flow rates, and in static and stopflow modalities. results: in this work, we focus on the specific capture of evs under different flow conditions to achieve an optimization of the incubation time. indeed, through the acquisition of real time binding data, we are able to precisely monitor the equilibrium point of the capture reaction. in this configuration of iris, low magnification optics allow for simultaneous detection of binding on hundreds of capture ligand spots. therefore, surface probes (surface density and specificity) as well as assay conditions can be optimized. we report on the optimization of antibodies against cd9, cd81, and cd63 markers. since the sensor chips are identical to the single-particle detection assays developed by nanoview biosciences, the optimization of binding assays will directly impact the phenotyping of individual exosomes. summary/conclusion: our method proved to be very efficient in optimizing the most crucial aspects concerning evs captureflow conditions, incubation time, surface density and sample concentration. introduction: diabetes is a life treating diseases extending its impairing influence on more than 500 billion of people around the world within upcoming 20 years. the most harmful complication generating high treatment and social costs is diabetic nephropathy, which develops in about 10% of patients suffering diabetes. still we do not have an effective and direct prognostic biomarker to diagnose renal complications in the primary stage of renal disease. methods: extracellular vesicles were concentrated from diabetic patients' urine and washed to perform spectral analysis: fourier transform infrared spectroscopy (ftir), based on the molecular absorption of electromagnetic radiation in the infrared region of the spectrum in a range from 400 cm-1 to 4000 cm-1 and raman spectroscopy (rs) as a technique based on inelastic scattering of monochromatic light. both techniques provide information on the chemical structure of compounds by identifying functional groups with high molecular specificity. results: average spectral signature obtained for evs from urine samples of patients in the different stage of kidney damage allowed distinguishing specific bands, representative for amide (i/ii), lipids, cholesterol and nucleic acids. spectral parameters correlated with a clinical stage and a commonly used indicator of renal function (creatinine) in diabetic patients. summary/conclusion: infrared and raman spectroscopy are promising tools to diagnose and monitor renal function in diabetes. introduction: several existing bioanalytical strategies for purifying and characterizing exosomes have allowed for fundamental progress to be made. mixtures of evs can be enriched for exosomes by techniques such as ultracentrifugation and size-exclusion chromatography. but, these processes require large amounts of material that are often difficult to obtain and many different types of particles have similar sizes and densities. it is likely that unique subfractions within enriched samples exist, particularly in complex biological matrices such as blood, urine or milk which remain difficult to characterize and isolate with existing analytical technologies. methods: bovine milk exosomes were isolated via differential ultracentrifugation and resolubilized in 100 mm ammonium acetate. these data were recorded using charge detection mass spectrometry (cdms). in cdms, individual particles are reflected back and forth through an electrostatic ion trap where they pass through a sensitive charge detector. each time a trapped particle enters and exits the detector, its charge (z) and mass-to-charge (m/z) ratio is measured. mass distributions are generated by multiplying the m/z values by the charge measured for each ion and binning the resulting masses.results: the masses of particles in a bovine milk extracellular vesicle (ev) preparation enriched for exosomes were directly determined for the first time by cdms. particle masses and charges span a wide range from m~2 to~90 mda and z~50 to~1300 e and are highly dependent upon the conditions used to extract and isolate the evs. in total, 57,350 particles were detected from eight cdms measurements. a simple two-dimensional gaussian model suggests that eight unique subpopulations of particles may be resolvable based on charge and mass. complementary em and proteomics analyses confirm that samples are enriched for exosomes. particles associated with the s1, s2, and s3 families that are centred at~3.5,~5.9, and~8.3 mda, respectively, appear too small to be ascribed to exosomes. the remaining 45,229 (79%) particles detected by cdms are within the mass range expected for exosomes. while cdms measurements are at an early stage of development, this approach appears to provide a new physical basis for separating and characterizing ev particles. summary/conclusion: this work describes a novel biophysical approach for measuring and characterizing the masses and charges of the extremely heterogenous population of exosomes and other extracellular particles enriched in bovine milk. as new sample preparation methods, aimed at purifying specific types of exosomes from different cell lines, tissues, and other body fluids continue to evolve, rapid and sensitive cdms measurements of the physical properties of mass and charge may become an important means of assessing the efficacy of different protocols. funding: nih (r01 gm131100-01). bab is supported by indiana university quantitative chemical biology fellowship (t32gm131994). in situ detection of exosomal microrna-10b by fusion with liposomeencapsulated nanomotor introduction: breast cancer is the most common cause of cancer-associated death in women and has raised global health concerns. early diagnosis and treatment are crucial to improve the prognosis and survival rate of breast cancer patients. liquid biopsy is expected to provide a strategy for early diagnosis of breast cancer. exosomes have been regarded as novel liquid biopsy biomarkers due to their stable cargo of rnas, lipids, and proteins from their origin cells. exosomal micro (mi)rnas have recently been recognized as promising indicators of cancer occurrence and progression. however, most of the reported exosomal mirna detection methods require the lysis or extraction process, which increases the possibility of sample loss. in situ detection strategies avoid interference from body fluid. in this study, we developed a gold nanomotor fluorescence platform based on liposome fusion for breast cancer exosomal mirna in situ detection. the exosomal mirna detection platform was constructed using a gold nanomotor (detector) and liposomes (carrier). the dnazyme amplification sequences which could be especially triggered by mirna-10b were identified by sds-page before modified on gold nano-motor and the capacity of the nanomotor was assessed using synthetic target sequence, breast cancer cell mda-mb-231, mirna-10b-encapsulated anionic liposomes, and mirna-10b-expressing exosomes. three kinds of liposomes were synthesized, characterized, and assessed for loading ability. membrane fusion effect was evaluated by confocal laser scanning microscopy (clsm) and nanoflow cytometry. the performance of this method to discriminate between breast cancer patients and healthy individuals was investigated. results: the chosen dnazyme amplification sequences transformed "locked" status to "cleavable" status on target addition, releasing a fluorescence signal. the modified gold nanomotor showed a ten times higher fluorescence signal in the presence of mirna-10b than the background and no noticeable fluorescence changes from a single-base-mismatch sequence. moreover, among the three different liposomes, cationic liposomes exhibited great stability, high loading efficiency, and excellent membrane fusion effect. furthermore, the fluorescent experiments confirmed that cationic liposomes could load and transfer the nanomotors into exosomes for mirna-10b detection. finally, we were able to distinguish breast cancer patients and healthy individuals by sensing exosomal mirna-10b directly from plasma samples without exosome isolation. summary/conclusion: a separation-free and sensitive assay based on dnazyme amplification technique and membrane fusion effect was established for breast cancer-derived exosomal mirna-10b detection, which could be a promising tool for the liquid biopsy of breast cancer. isolation of exosomes by membrane affinity column increases non-exosomal rna recovery in comparison to differential ultracentrifugation introduction: exosome-based liquid biopsy is a potential aid in the diagnosis and prognosis of cancer patients. however, in order to incorporate exosomes into clinical routine, there is a need to compare different isolation methods. here we analysed the impact, in exosomal rna yield, of two intermediate recovery/ intermediate specificity methods: differential ultracentrifugation (ucd) and a membrane-affinity column (mac) kit. although mac has a faster performance which is more suitable to the clinic, we found that ucd results in a higher recovery of exosomes and less contaminating non-exosomal rna.methods: exosomes were enriched by mac and ucd from identical volumes of human plasma (12,000xg, 34 min/0.22 ) m filtration/110,000xg, 5 h)(n = 7) and lymphoma conditioned medium(300xg, 10 min/200xg, 20 min/1000xg, 30 min/120,000xg 1.5 h/120,000xg, 1 h) (n = 3). all exosomes were characterized by nanoparticle tracking analysis (nta), immunoblotting of cd63/cd9/flotilin/alix and electron microscopy (tem). exosome pellets were pre-treated with proteinase k (1 mg/ml/56°c/10 min) and rnase a (2 mg/ ml/37°c/20 min) before phenol-chloroform/glycogen rna extraction. rna yield was measured by both fluorometer and bioanalyzer.results: isolation of exosomes by ucd, in both plasma and medium, resulted in a higher yield in comparison to mac. this was shown by an augmented intensity of marker bands in the ucd samples (p = 0.005, n = 4) as well as by an increased number of exosomes in tem.in contrast, mac final exosomal fraction (from both plasma and medium), resulted in a 13-fold and 200fold increase in rna, respectively, in comparison to ucd when measured by fluorometer. this was confirmed by bioanalyzer. introduction: there is a need for better techniques for characterizing ev populations. we developed a sensitive multiplexed electrochemiluminescence (ecl)based assay format to characterize evs in cell-conditioned medium (ccm) and human biofluids. here we use the format to analyse ev samples for the presence of 66 ev surface proteins, and to identify changes in ev phenotype associated with different cell lines, purification methods and growth conditions. methods: multiplex plates were prepared on msd's u-plex® platform with antibodies for 66 putative evsurface proteins. each well displayed an array of nine specific capture antibodies and a negative control antibody. evs from samples were captured on the arrays and then detected with a cocktail of anti-tetraspanin antibodies (cd9, cd63 and cd81) conjugated to an ecl label. three distinct cell types were grown at two sites, msd and atcc. resulting ccm were each purified by four common methods: tangential flow filtration, peg-based precipitation, size-exclusion chromatography and centrifugal ultrafiltration. all samples were also assayed without purification.results: fifty-five of the surface markers were detected on intact evs from at least one evaluated cell type. datasets were analysed using correlation matrices, hierarchical clustering, and machine learning. for each cell type, when comparing unpurified ccm grown at different sites or evs prepared by different purification methods, we typically observed correlations above 0.9, indicating that the purification methods did not introduce bias to ev phenotypes, and that the assay format can provide robust phenotypic information without any purification of evs. two unsupervised clustering analyseshierarchical clustering and t-distributed stochastic neighbour embeddingboth generated wellseparated clusters for each of the cell types, regardless of purification method or source. summary/conclusion: we developed multiplex ev surface marker assays and demonstrated their use for multimarker ev phenotyping. this flexible format enables rapid assay development for new ev subpopulations with or without sample purification. these results also demonstrate ev surface marker phenotyping via multiplex ecl assays may be used to distinguish ev populations from various cell types, and characterize bias introduced by purification. detection of misev recommended ev protein-markers using automated western blotting method for isolation of evs and a simple western blotting platform for automated protein separation and immunodetection of misev-recommended proteins.methods: total evs were isolated by affinity-membrane spin columns from pre-filtered 0.5-4 ml plasma or 2-20 ml urine, respectively. intact vesicles were eluted and the ev-depleted biofluid fraction was collected from the flow-through. a small fraction (4 μl) was analysed by a simple western blot workflow providing automated capillary electrophoresis-based protein separation and immunodetection, characterizing each fraction for presence or absence of misevrecommended proteins.results: a range of specific antibodies were identified and the ev fractions were shown to be enriched in evproteins, whereas contaminating non-ev proteins were significantly reduced. isolation of evs was necessary to allow detection of the low abundant ev protein markers, whereas non-ev proteins were readily detectable both in the neat biofluids and in the ev-depleted flowthrough. we characterized the effect of washing on the purity of ev isolates and defined the dynamic range of the workflow using titrations of input volume of both plasma and urine ev isolations. summary/conclusion: simple western blotting protocols were established for quality control of isolated evs in accordance with misev-guidelines. evs isolated using affinity-membrane spin columns were shown to be enriched in ev markers and depleted for non-ev proteins. al-pha beads: a library of extracellular vesicle-associated metalloproteinase biosensors (adams) and a disintegrin and metalloproteinase with thrombospondin motifs (adamtss) are highly promising cancer biomarker candidates that have complex roles in cancer pathogenesis and metastasis. importantly, within the context of lung cancer, the detection of adam10 proteolytic activity might be more informative than the level of adam10 protein.therefore, the development of low-cost metalloproteinase biosensors could serve as useful biomarker research tools. methods: to this end, we developed advanced proteolytic detector polyhydroxyalkanoates (al-pha) beadsa library of biodegradable, biopolymer-based protease biosensors. broadly, these biosensors utilise phac-reporter fusion proteins that are bound to microbially manufactured bioplastic beads. these phac-fusions also incorporate specific protease cleavage sites. in the presence of a specific protease, reporter proteins are cleaved off of the al-pha beadsresulting in a loss of bead fluorescence that can be measured using flow cytometry. these biosensors were assayed using either metalloproteinases, conditioned media or evs from in vitro cancer models.results: human metalloproteinase recognition motifs were identified in the literature and a total of 70 different al-pha bead biosensors were designed. a control, tev-specific biosensor detected 0. introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr.results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom20. mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the 374 isev2020 abstract book biogenesis of evs is neutral sphingomyelinase 2 (nsmase2), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase2 is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase2 inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles.methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo [1,2-b] pyridazin-8-yl) pyrrolidin-3-yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated 5xfad mice with 10 mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay.results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, 5xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting 20% of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human postmortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of 20 mdd subjects and 20 hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed.results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd9. tem images showed the typical cup-shaped morphology with sizes mostly between 30 and 200 nm. preliminary sequencing results revealed that mir-33a-5p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir-33a-5p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. methods: we use ifc to characterize evs released by glioma using 5-ala, fluorescently labelled ev (cfda-se, cd81) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd81) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that 58% are tenascin c positive, 2.7% are egfrviii positive and 1.6% are 5-ala positive. there was only a minor overlap (<16%) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs (2.5-fold), tumour specific evs (2.8-fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll 2 receptor (tlr2) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il-12 and il-6, which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells.objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow field-flow fractionation (af4) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp-1) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk-2 epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with 50 to 50 nm (ev1) and another with 100 to 120 nm (ev2). ev1 induced more tnf-alpha, il-6, ip-10 and ccl20 than ev2. it was also more effective in promoting t. cruzi infection in epithelial cells. due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays.noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr.isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and 0.2um filtration. co-purification of norovirus with the evs was checked.ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid50 assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems.ps15.12 = op3.12 kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of 11.5 µg/ml of plasma, as geometric means ≥11.5 µg/ml were nearly equal. hevs were detected at 48 µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f200x electron microscope and measured between 40-90 nm, and hevs were between 60-160 nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: nasopharyngeal carcinoma (npc) is characterized by a large presence of regulatory t cells (tregs) and the production of tumour-derived exosomes with immunosuppressive properties. our team showed that npc-derived exosomes favour the suppressive activity and recruitment of human tregs via ccl20 chemokine, thus contributing to npc immune escape (mrizak et al., jnci, 2015) . more recently, our team has shown that npc-exosomes could induce tregs by altering the maturation of dendritic cells (dcs) and promoting tolerogenic dendritic cells (tdcs) (renaud et al., herpas congress 2017). our main objectives in this study are (i) to define and compare the metabolic status of mature dendritic cells (mdcs), control tdcs and tdcs generated in the presence of npc-exosomes (exocnptdc) and (ii) to evaluate the chemoattractive potential of npc-exosomes on exocnptdcs, and notably to investigate the involvement of ccl20 in this recruitment. methods: dcs are generated from human monocytes in the presence or absence of npc-exosomes. the maturation status of dcs was evaluated at a phenotypic level by studying the expression of maturation markers using flow cytometry and at a functional level by analysing cytokines secretion using elisa. this cytokine analyse has been performed in both conditions, on treated dcs and during co-culture assays of autologous cd3 t lymphocytes with treated dcs. in a second step, a mitochondrial metabolic and glycolytic study was performed using the seahorse technology (ocr and ecar measurement). finally, the chemoattractive potential of npc-derived exosomes on the different induced dcs was analysed (i) using boyden chamber chemoattraction assays or real-time videomicroscopy (chemotaxis µslide ibidi) and (ii) using rt-qpcr analysis of the receptor expression of ccl20 (ccr6).results: npc-exosomes alter dc maturation, which gives rise to tolerogenic dcs that favour the induction of tregs. in addition, the metabolic analysis of dcs seems to put foward a specific metabolic signature of the tdcs induced by npc-exosomes. and finally, chemoattraction assay suggests that npc-exosomes preferentially attract tdcs and exocnptdcs in a ccl20dependant manner. summary/conclusion: taken together our results should allow us to characterize the major role of npc tumour exosomes on the maturation and the recruitment of dc and so identify them as anti-tumoural therapeutic targets. cytotoxic t lymphocyte ev that prevents tumour metastasis by collapse of tumoural mesenchymal stroma is classified into exosome, but not microvesicle or apoptotic body.naohiro seo a , junko nakamura a , tsuguhiro kaneda a , takanori ichiki b , asako shimoda c , kazunari akiyoshi c and hiroshi shiku a a mie university graduate school of medicine, mie, japan; b the university of tokyo, bunkyo, japan; c kyoto university, kyoto, japanintroduction: recently, instead of ultracentrifugation, development of new preparation protocol is demanded for research of reliable bioactivity and drug discovery of extracellular vesicles (evs). in this study, we propose a novel method for large scale preparation of highperformance extracellular vesicles focusing on membrane negative charge. methods: murine cytotoxic t lymphocyte (ctl) evs in supernatant were concentrated more than 20 times at over 97% purity without leaking by 750 kda mwco ultrafiltration, and subjected to ion exchange deae column chromatography after replacing with pbs. after ion exchange, evs were characterized by bca assay, nta assay, cryotem observation, proteome analysis, dna content measurement, mirna microarray analysis, zeta potential measurement, lectin array analysis, and target cell analysis.biomarker detection and analysis and detail strategies for cross-platform analytical validation. methods: we conducted a cross-platform analysis using two commercially available flow cytometers designed for ev detection. scatter resolution, enumeration accuracy and precision were determined across both platforms by analysing submicron silica beads (apogeemix, 180-1300 nm) of known concentration.we detected large evs, as established by reference size beads, electron microscopy, expression of phosphatidylserine and the presence of integral membrane proteins of cell of origin. we analysed evs isolated from plasma by high-speed centrifugation (18,900 g) as well performing analysis by direct plasma labelling followed by validation by detergent lysis of vesicular constituents. a clinical operating range was defined which ensures linearity and avoids swarm detection. we observed comparable scatter resolution, enumeration accuracy (error ≤15%) and precision (cv ≤5%) across both platforms used. we defined two ev size gates: a "latex" gate (300 to 1100 nm polystyrene latex beads), and a "silica" gate (180 to 1300 nm silica beads) for evs at the lower end of our size range of interest. to improve detection sensitivity, we identified common contributors to signal noise and applied workflow strategies to minimize these. finally, we identified linear ranges which avoid swarm detection, and which ensures reproducible ev counts (cv <20%) across both instruments. summary/conclusion: we present an optimised, standardised and cross-platform reproducible working protocol which supports the use of fcm in an ev-based liquid biopsy application. funding: the project is funded by spark oceania and uts innovation commercialisation seed fund scheme to mb. metabolomic profiling of serum and exosomes isolated from head and neck cancer patients after radiotherapy introduction: cancer radiotherapy (rt) induces the response of the whole body that could be detected at the blood level. searching for new molecular signatures which could correlate with treatment response in cancer patients is of particular importance. radiation-induced changes in proteome and transcriptome of serum have been widely described. however, metabolomic changes in serum, exosomes and other classes of small extracellular vesicles (ev) of cancer patients after rt have not been given as much attention. metabolomics of serum and ev of cancer patients could provide a valuable insight into the response of both tumour and whole organism to the treatment. the aim of the study was to compare serum and ev metabolomic profiles in head and neck cancer (hnc) patients before and after rt. methods: serum samples from 10 hnc patients were taken before (a) and after (b) rt. 10 healthy volunteers were used as a control group (c). ev were isolated from 1 ml of serum using size-exclusion chromatography (sec). selected sec fractions were subjected to extraction of metabolites. a mixture of meoh/h2o was used for extraction of metabolites from serum and ev samples. samples were analysed by gas chromatography-mass spectrometry (gc-ms).the study protocol adhered to the tenets of the declaration of helsinki and was approved by the bioethical committee of the maria skłodowska-curie national research institute of oncology, branch gliwice, poland (permit nr. do/dgp/493/4/06/1/2016/g). results: an untargeted gc-ms-based approach allowed the detection of 189 metabolites in serum samples and 50 exosomal small molecules, of which 32 joint. the identified compounds included amino acids, fatty acids, carboxylic acids, sugars, and others. there were 49 metabolites which levels discriminated compared groups (a,b,c) of serum samples and 12 compounds that discriminate the ev isolated from hnc serum before and after rt from hc. summary/conclusion: rt caused significant changes in levels of serum and ev metabolites witch are involved in amino acid metabolism, lipids metabolism, energy metabolism and oxidative stress response. capable of contributing to intercellular communication and metastasis. numerous studies have focused on elucidating their role in cancer progression. we recently showed that sevs isolated from pancreatic cancer cells can function as an initiator in malignant cell transformation. here, using a mass spectrometry (ms)-based proteomics approach, we analysed the differences in the protein cargo of sevs secreted from normal pancreatic and cancer cells to better understand their biological characteristics. methods: sevs were isolated from 3 human pancreatic cancer cell lines (capan-2, mia paca-2, and panc-1) and normal pancreatic epithelial cells (hpde) using a combined ultrafiltration-ultracentrifugation method coupled with a sucrose density gradient purification. proteomic profiling of sevs was carried out using an lc-ms/ms method. protein identification from resulting ms/ms spectra was conducted using proteome database search software followed by gene ontology (go) enrichment and reactome pathway analysis.results: a total of 4,907 unique proteins were identified confidently across the combined samples. the proteins present in all four sev types (1,135 proteins) consist of general housekeeping proteins. 348 proteins were uniquely found in all cancer sevs but not in the normal hpde sevs. this group contains an enrichment of proteins that function in the endosomal compartment of cells responsible for vesicle formation and secretion and suggest their important role in driving the increased production of sevs from cancer cells relative to normal cells. moreover, this group includes a set of proteins that have been implicated in malignant cell transformation, consistent with our previous work showing that each of the cancer sevs analysed here could initiate malignant transformation of nih/3 t3 cells. conversely, there were 313 proteins uniquely found in normal hpde sevs. this group includes a number of immune response proteins that are not found in any of the pancreatic cancer cell sevs. summary/conclusion: the differences in the proteomes of cancer and normal sevs may be indicative of their varying roles in cell transformation and helpful in delineating the types of evs that are being produced. in addition, these differences point towards their potential value as cancer biomarkers. proteomic profile of tumour-derived exosomes in plasma of melanoma patients introduction: in the past years, extracellular vesicles (evs) have attracted considerable interest due to their ability to provide valuable diagnostic information from liquid biopsies. the high abundance in all bodily fluids and their cargo stability confers evs the potential as a powerful tool to not only obtain novel biomarkers from inaccessible tissues, therapy response and monitoring, but also to reduce infection risks of conventional highly invasive biopsies. virtually all cells continuously release vesicles into the extracellular environment, diverse in size, content and features depending on the biogenesis, origin and function. this heterogeneity adds a layer of complexity when attempting to isolate and characterize tissue-specific vesicles. methods: hence, we aimed to use a immunomagnetic capture approach for prostate-derived evs from cell culture supernatants, with further investigation into human plasma and urine samples. analysis was performed by nanoparticle tracking analysis, western blotting and electron microscopy. additionally, an in-house spotted antibody microarray is in development. here, we intend to detect different ev sub-populations based on their surface markers. results: isolated immunocaptured ev populations based on the classical ev marker cd9 show an increased signal for the luminal protein tsg101. ev populations targeting the tissue-specific marker prostate specific membrane antigen (psma), were found positive for tsg101 in a lower extent indicating a subpopulation of evs. the microarray uses less than 100 µl of sample (concentrated cell culture supernatant, human plasma, urine) and leads to a faster characterization within 3 h for ev surface marker as compared to western blot. summary/conclusion: immunomagnetic isolation might be a promising approach for liquid biopsy and thereby the microarray could be valuable to identify potential capture targets. the current design for 6 different surface marker from 16 samples simultaneously could be easily extended for sample size and surface profiling allowing for a more economical way to multiplex samples. paving the way for implementing a feasible and reliable technique for assessing urinary extracellular vesicles as biomarkers for bladder cancer in clinical practice introduction: extracellular vesicles (ev) in urine have been proposed as biomarkers for bladder cancer (bc). however, at present there are no standardized methods for ev isolation or urine sampling. our goal was to evaluate the ev isolation performance between different methods, the effect of the sampling time and the importance of urinary creatinine (ucr) normalization. methods: two urine samples of 120 ml were collected from 5 patients with non muscle-invasive bc: one from the first micturition and another from any time of the day. twenty ml were used for ucr measurement and 100 ml were used for ev isolation by either precipitation with polyethylene glycol (peg), concentration by filtration (uf, centricon plus-70, 10 k, millipore), sepharose size exclusion column (sec), or combinations of these methods. additionally, the effect of protease inhibitors (pi) and dtt treatment after collection or during processing was analysed. size and number of particles were evaluated by nanosight and the presence of exosomal markers was evaluated by western blot. results: among the methods evaluated, uf + sec showed the best performance retrieving the highest number of particles in the range of 50-200 nm, and the highest protein expression of exosomal proteins. uf alone showed the highest concentration of ev, but with a tendency to isolate larger particles. particle concentration was positively correlated with ucr, reflecting the importance of ucr normalization before journal of extracellular vesicles 385 comparing between patients. finally, no differences in the performance according to the time of collection, nor in the use of pi or dtt were observed. summary/conclusion: uf + sec gave the highest ev yield and was not affected by the time of urine collection. the use of pi and dtt can be avoided, and normalization to ucr should be considered when implementing this technique for assessing evs as biomarkers for bc in clinical practice. funding: pida 2019. the introduction: human tumours, including pancreatic ductal adenocarcinoma (pdac), often harbour a subpopulation of cancer cells with extra centrosomes. we found, that these cells secrete an increased number of small extracellular vesicles (sevs), within the 20-120 nm size range. sevs play a role in cancer signalling and progression and are widely studied for their diagnostic potential. we aim to understand the role of sevs secreted by cells with extra centrosomes in shaping pdac-associated stroma, particularly fibrosis. methods: to study the sev mediated changes in the pdac microenvironment, we purified sevs through serial ultracentrifugation and size exclusion chromatography, characterised the content through silac-based proteomics, and assessed phenotypic changes in pancreatic stellate cells (pscs) and extracellular matrix (ecm) production through immunofluorescence staining. results: our data indicates, that the sevs secreted by cells with extra centrosomes are exosomes due to their endocytic origin, and we found, that they can activate pscs, key mediators of fibrosis in pdac. indeed, we observed an increased level of collagen i produced by pscs activated by sevs from cells with extra centrosomes as compared to cells without extra centrosomes. interestingly, we found, that psc activation through sevs is not mediated by tgf-β, assessed by the level of nuclear smad2 accumulation downstream of tgfβ activation, suggesting a novel mechanism of pscs activation. summary/conclusion: pdac cells with extra centrosomes contribute to a novel type of psc reprogramming, which could alter their ecm deposition and contribute to the extensive fibrosis observed in pdac. we are currently characterising the signalling pathways associated with sev mediated psc activation and how it impacts padc progression to better understand the role of centrosome amplification in the cancer-stromal crosstalk. exosomal carboxypeptidase e confers and cpe-shrna loaded exosomes inhibit growth and invasion of hepatocellular carcinoma cells. methods: exosomes were isolated from the culture media of high metastic hcc97 h cells and incubated with low metastatic hcc97 l cells. in other experiments, cpe-shna loaded exosomes from hek293cells were incubated with hcc97 h cells. the recipient cells were analysed for proliferation using mtt assay, colony formation, and matrigel invasion. results: analysis of exosomes derived from hcc97 h cells revealed cpe-wt mrna and protein. exosomes released from hcc97 h cells were able to enhance proliferation and invasion of hcc97 l cells. when cpe expression was suppressed in the hcc97 h cells before exosome isolation, the exosomes had no effect on proliferation and invasion. these data demonstrate the ability of exosomes to confer growth and invasion in hcc cells and the role of exosomal cpe in driving the process.previously it was shown that down-regulation of cpe expression by shrna can reverse tumour growth and metastasis in an hcc mouse model. we therefore loaded cpe-shrna into exosomes by infecting hek293 (human embryonic kidney) cells with adenovirus carrying cpe-shrna-gfp. these modified 386 isev2020 abstract book exosomes were used to transfer cpe-shrna to hcc97 h cells, resulting in significant reduction in proliferation and colony-forming ability of these cells. cpe-shrna loaded exosomes were found to down-regulate the expression of cyclin d1 and c-myc, two genes with high relavance to tumour growth and metastasis. summary/conclusion: our results demonstrate the ability of exosomal cpe to enhance proliferation and invasion in low metastatic hcc cells and the potential to use shrna loaded exosomes to target cpe as a therapeutic strategy to treat liver cancer.funding: intramural program of the eunice kennedy shriver national institute of child health and human development, and national cancer institute, national institutes of health, bethesda, md. 20892. stress hormones promote prostate cancer aggressiveness through modulation of mir-628-5p expression and exosome release north carolina central university, durham, usa introduction: despite proactive screening and steady declines in mortality, prostate cancer (pca) remains one of the most prevalent cancers among men. evidence suggests that chronic activation of stress signalling pathways can result in an altered mirnas transcriptome and affect exosomal content and release. here, we study the interaction between leptin and mir-628-5p expression, previously shown to be downregulated in pca patients. in addition, explored the effect of stress hormones cortisol and leptin on exosomal release and content from pca cells.methods: we utilized normal prostate cell line rwpe-1, and pca cells pc3, lncap and mda-pca-2b. proliferation of cells treated with leptin in the presence or absence of mir-628-5p mimic or negative control was assessed by mtt, colony formation, wound healing, and expression of targets affected by mir-628-5p was assessed by western blotting. moreover, exosomes were isolated via differential centrifugation from pca cells treated with leptin or cortisol and exosome number was determined by nanotracking analysis. exosome content was determined by western blotting and proteomic analysis by mass spectrometry.results: we observed that leptin significantly decreased expression of mir-628-5p in rwpe-1 cells.co-treatment with mir-628-5p mimic and leptin abrogated these effects in a cell dependent manner. we also observed that co-treatment with leptin affected mir-628-5p target jag1 and other molecules involved in epithelial to mesenchymal transition. in parallel, we demonstrated that cortisol increases exosome secretion particularly in pc3 cell exosomes with a 2.6-fold increase at 5 nm cortisol compared to untreated. western blotting revealed the presence of gr in exosomes particularly at 5 nm cortisol. summary/conclusion: understanding epigenetic regulation through mirnas and exosomes may be the key to understand stress hormone influence in pca progression. these findings suggest that stress hormones effectively affect mir-628-5p expression and exosomal release and signalling.introduction: extracellular vesicles (evs) are promising drug delivery vehicles for therapeutic microrna (mirna). for the loading of exogenous cargo, researchers broadly seek to either manipulate the evs directly or the cell that produce them. electroporation, sonication, and direct ev transfection are common methods that work by physical disruption or irreversible chemical addition, which may irreparably damage the molecules intended for therapy. on the other hand, transfection into the producer cells is a simple option that does not imperil ev integrity.methods: there are multiple factors that contribute to ev loading efficiency, including transfection reagent used, timing, and dosage. thus, we sought to establish a basic protocol and improve understanding of the underlying dynamics involved in a basic system consisting of hek293 t cells and mir-146a-5p mimic.results: in this work, we examined how different reagents lead to variable ev loading. then we looked at variable dosages, specifically the relationship between rna amount added to reagent, amount present in cell, and amount exported to evs. summary/conclusion: these results will help future studies produce evs with exogenously loaded small rna, and suggest future optimizations. funding: national institutes of health. r01 and t32 (host pathogen interactions at university of maryland). we report a single ev trapping method via aptamermediated assembly between au nanoparticle (aunp) and au superlattice template. we propose a chip-based ev trapping technique based on semiconductor processes. methods: we introduce aptamer coated au nanoparticle (aunp) and au superlattices as a template to capture evs. first, we fabricated poly(methyl methacrylate) (pmma) hole pattern on au-coated si substrates by using electron beam lithography (ebl). we designed 200 nm-diameter hole patterns to capture one ev in each hole. to connect the aunp and the au superlattice template, we used an aptamer molecule as a linker strand. also, to capture individual evs, the aptamer molecule is designed to have a hairpin structure to specifically bind to cd63, a protein marker of ev. we modified 5ʹ-terminal and 3ʹ-terminal of the cd63 aptamer with thiol group for the formation of self-assembly monolayer (sam) on both aunp and au superlattice surface. results: first, we coat the cd63 aptamer on the surface of aunp. afterwards, we load the aptamer-coated aunp into au superlattice template. ev solution is specifically bound to cd63 aptamer. after washing step, each ev is expected to locate within a single hole due to the size confinement of the hole. to separate the evs from the aptamer, we use restriction enzyme, bamhi, to recognize specific dna sequence and cleave them. summary/conclusion: in this report, we propose a aunp -linked au superlattice chip by aptamer molecules for trapping evs. we selected cd63 aptamer for specifically binding with cd63 in evs. in addition, we designed cd63 aptamer as a linker strand to connect introduction: a hallmark of platelet activation is the release of internal granules as extracellular vesicles/ microparticles. thrombolux is a dynamic-light-scattering-based (dls) instrument that was developed for use in clinical setting to check for platelet activation before transfusion. compared to traditional dls, the thrombolux requires no cleaning (single-use capillary) and requires very little sample (70 µl). hence the thrombolux may be a useful instrument beyond platelet pack test in blood transfusion laboratory. we have evaluated its use as an in-process monitoring tool for industrial ev manufacturing, for both quantifying cells (input) and evs (output). methods: the thrombolux was used to test the activation status of expired platelet packs (donated by arcbs for research purpose). the readout was compared with platelet swirling test and flow cytometry data (surface marker). furthermore, the thrombolux was also tested for process development and ev manufacturing monitoring purposes at different stages of the process for its ability to rapidly obtain particle presence and size information on evs. time to result was also compared between different particle analysis methods. results: the thrombolux was a better predictor of platelet packs variability compared to the traditional platelet swirling method. however, we did not observe a strong correlation between the activation status and the flow cytometry-based activation marker data. the thrombolux was able to provide a useful estimation of particle presence and sizing of evs in-process.results are obtained rapidly, within minutes, with minimal sample prep. summary/conclusion: although we did not observe a significant direct correlation between flow cytometry activation data and the % microparticles (within a small sample size), the thrombolux has shown potential to become a useful tool for in-process monitoring for ev manufacturing and other ev research, in particular through its speed and ease of use. funding: all funding was through exopharm ltd (asx:ex1). secreted introduction: a major manufacturing challenge related to exosome bioprocessing is that of robust and scalable purification. as efforts to translate exosomes into clinics grows, the more important the design of quality systems which can reproducibly purify the product becomes. the current gold-standard, ultracentrifugation, was adopted from the viral vaccine industry, but remains imperfect in terms of scale up and manufacturing due to labour and time intensive process requirements. in order to follow the preferential adoption of more standard bioprocesses, as previously achieved by the viral vaccine industry, we show the development of two monolith chromatography steps which can be used to purify exosomes from a clinically relevant, allogeneic stem cell product (ctx0e03). methods: t-flask expansion of ctx0e03 cells was performed to yield batches of 5-15 l of conditioned medium. the medium was subsequently clarified by benchtop centrifugation, and concentrated into a crude concentrate by tangential flow filtration [tff], using a combination of 0.22 µm dead-end filtration prior to concentration in a 300kda hollow-fibre tff system. tff retentate was loaded onto 1 ml hic or aex monoliths, for further purification. potency was assessed by a fibroblast wound healing assay in vitro. results: exosome presence was verified in the tff material by detection of cd 81 and cd 63. exosomes recovered in this manner could achieve full wound closure in vitro over 72 hours, when dosed at 20 µg. further purification by monolith chromatography showed high levels of reduction of albumin, detected by western blot, as well as heightened ratios of particles to both total protein, and total dna. the results indicate that neither aex nor hic steps cause detrimental loss to product function, either alone or in combination with one another. introduction: custom-made platelet pellet lysate (ppl) and heat-treated ppl (hppl) exert strong neuroprotective effects of neurotoxin-exposed dopaminergic luhmes neuronal cell culture. this effect is significantly enhanced using hppl, which was also highly protective of th-expressing neurons in mice parkinson's disease (pd) model. introduction: there is a critical unmet medical need for new therapies to treat age-related diseases including cardiovascular diseases such as stroke. exosome derived from stem cells have shown intrinsic therapeutic potential in a variety of animal models of ischaemic diseases. we have identified scalable exosome production cell lines (purestem) as a source of angiogenic exosomes and are aiming to generate good manufacturing practice (gmp) grade therapeutic exosomes that can effectively mediate angiogenesis and tissue regeneration. we are developing exosome production and purification protocols that combine methods of tangential filtration flow (tff) and size exclusion chromatography (sec). the particle number and size were measured by both tunable resistive pulse sensing (trps) as well as nanoparticle tracking analysis (nta) for comparison. exosomes were characterized by detection of exosome surface markers and absence of cellular markers. purity was assessed by measuring particles per ug of total protein content. the angiogenic activity of purestem-exosomes was assessed using live-cell imaging to measure endothelial wound-healing and tube formation assays. we further investigated the molecular cargo of purestem-exosomes by screening mirnas targets, rna-seq analysis, and mass spectrometry analysis.results: the isolated purestem-exosomes using our developed protocols were highly purified, resulting purity in the range of 1e10-5e10 particles/ug. we selected angiogenic exosome-producing cell lines from our purestem library by screening for functional activity and characterizing their molecular cargo. we found that purestem progenitor-derived exosomes showed higher angiogenic potency than primary mesenchymal stem cell (msc)-derived exosomes. furthermore, angiogenic micrornas such as mir-126 were enriched in purestem-exosomes from certain producer cell lines. summary/conclusion: these data demonstrate the potential for using purestem lines as a highly scalable source of therapeutic exosomes. we were able to obtain highly pure exosomes that retain their angiogenic activity. we anticipate that purestem-exosomes will be a valuable resource for developing ev therapies for stroke and other ischaemic diseases. we have developed purification methodologies aimed at achieving a robust and scalable exosome production compatible with gmp for clinical grade purestem-exosomes. these developments have great potential as therapeutic agents for future preclinical in animal model of stroke and clinical trials. neuronal introduction: the hallmark of parkinson's disease (pd) is a-synuclein accumulation, predominantly in dopaminergic neurons, causing neurodegeneration. pd is also associated with insulin resistance, a condition characterized by phosphorylated insulin receptor substrate-1 (irs-1). besides motor symptoms, some pd patients develop mild cognitive impairment (pd-mci) or dementia (pd-d). given the importance for prognosis, there is an urgent need to develop biomarkers for distinguishing pd with normal cognition (pd-n) from pd-mci/d. neuronal-origin extracellular vesicles (nevs) contain cell signalling and pathogenic proteins (including a-synuclein), which may serve as biomarkers for alzheimer's disease, pd and other dementias.methods: from 0.5 ml of plasma from 104 pd-n, 83 pd-mci, and 39 pd-d patients, we immunocaptured nevs using anti-l1cam antibody. then, irs-1pser312 and irs-1ptyr20 and a-synuclein were measured in nevs using electrochemiluminescence immunoassays.results: a-synuclein was lower in pd-mci and pd-d compared to pd-n (p < 0.005) and significantly decreased with increasing motor symptom severity measured by mds-updrs iii score (p = 0.005). irs-1pser312 was lower in pd-d than in pd-n. irs-1ptyr20 significantly decreased with increasing mds-updrs iii score (p < 0.005). no biomarker was associated with disease duration. summary/conclusion: pd patients with cognitive impairment exhibited lower nev levels of a-synuclein than cognitively intact pd patients, whereas a-synuclein and irs-1ptyr20 were inversely associated with pd motor symptom severity. additional biomarkers and measurements will be available by the time of isev. plasma nevs is a valuable tool for discovering biomarkers in pd and investigating aspects of disease progression. introduction: despite decades-long advancement in transplant medicine, there is a necessity for personalized approach regarding early kidney allograft injury recognition and immunosuppression therapy towards improved transplant outcomes. biopsy, a gold standard for assessment of kidney allograft injury, cannot be serially used for the diagnosis of subclinical injury due to it's invasiveness and possible sampling errors. instead, urine is easily obtainable and bearing extracellular vesicles (evs), potential carriers of pathological signals related to kidney injury. our aim was to set up a urinary ev (uev) isolation protocol that would allow consistent and reliable identification of their characteristics and cargo. methods: second morning urine sample (25 ml) was collected from 7 patients and processed within 4 hours. oxalate precipitation, ph and dilution variability, uromodulin polymerization and high protein content were taken into account. isolated evs were defined by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). uev specific proteins and mirnas were analysed by western blot and qpcr, respectively. results: the optimal protocol relied on low speed urine centrifugation (2.000 x g, rt) for cell removal and storage at −80°c prior to further analyses. after urine thawing at rt, added edta averted cryoprecipitate and uromodulin polymer formation, while concentrated pbs neutralized the ph. filtration through 0.22 µm pores was used for large particle removal, while centrifugal 100 kda membrane units (amicon®, milipore) served for sample concentration followed by particle separation on sizeexclusion chromatography (sec; qevoriginal, izon q). protein vacant sec fractions (as rated at a280) were pooled and concentrated to a volume of 70 µl. tem micrographs revealed high sample purity and cup-shaped morphology of uevs. as per nta results, the average mean size of evs was 129,9 nm with concentration range of 1 × 109 particles/ml of starting urine. uevs were positive for the tested marker proteins hsc70, flotillin, tubulin, gadph and cd63. qpcr verified mirna presence in uevs, with ct for mir let-7i at 20. summary/conclusion: we successfully isolated pure uevs. the set up protocol will be used to assess uevs as non-invasive biomarkers of allograft injury in kidney transplant recipients. astrocyte-derived extracellular vesicles regulate dendritic spine formation and neuronal network connectivity introduction: recent advancements in the biology of extracellular vesicles have begun to implicate glial released microvesicles as mediators of glia to neuron communication, suggesting that alterations in the release and/or composition of astrocyte microvesicles could impact neuronal function. methods: astrocytes were allowed to constitutively release extracellular vesicles (adev-cr), or stimulated with atp (adev-atp). adevs were isolated by ultracentrifugation followed by proteomic analysis. we developed a normative whole transcriptome database using primary neurons exposed to adev-cr, and identified changes in neuronal gene expression produced by exposure of neurons to adev-atp. we identified a number of pathways associated with the biological response of synapse, spine and neurite outgrowth that were regulated by adev-atp. the molecular cargo of adev-atp responsible for regulating synaptic functions in neurons were characterized by biochemical, molecular, and functional assays. results: adev-atp enhanced the maturation of dendritic spines and produced functional enhancements in neuronal activity and network connectivity. the mechanism for this effect involved the delivery of integrin-4 1 and epha2 that were enriched in adev-atp. integrin-4 1 facilitated binding of adevs to the neuronal surface, and epha2-receptor signalled through ephrin to the tyrosine kinase erbb2/ 4 that regulated the phosphorylation and activation of trkb without increasing expression of the natural ligands bdnf or ntf3. this direct activation of trkb increased the expression of the synaptic scaffolding proteins disc1, arc, and cplx3 to promote the maturation of dendritic spines. this increase in mature dendritic spines was associated with increased neuronal activity and network connectivity demonstrating a functional strengthening of synapses. summary/conclusion: these data identify a molecular mechanism whereby modifications in adev protein cargo produced by the stimulation of astrocytes with atp regulates synaptic maturation through activation of trkb in a manner independent of growth factors. stephanie kronstadt and steven m. jay university of maryland, college park, college park, usa introduction: mesenchymal stem cell extracellular vesicles (msc-evs) have been shown to have an immunosuppressive effect in both autoimmune and inflammatory disorders. despite this, clinical translation of ev therapies is hindered by potentially low potency in vivo and the lack of a scalable biomanufacturing process. cell culture parameters are critical in modulating both yield and bioactivity of evs. thus, we hypothesized that the combination of chemical priming and 3d dynamic culture would enhance the yield and potency of immunosuppressive msc-evs. methods: bone marrow-derived mscs cultured in flasks were chemically primed using ethanol or curcumin. mscs were also cultured using a 3d-printed scaffold-perfusion bioreactor using a flow rate of 5 ml/min. anti-inflammatory effects were assessed following application of msc-evs to lipopolysaccharide (lps)-stimulated murine macrophages. subsequent inhibition of the production of the pro-inflammatory cytokine il-6, quantified using an elisa, was used to characterize evs as anti-inflammatory. in addition, both chemical priming and the bioreactor will be simultaneously utilized to potentially uncover any synergistic effects on ev immunomodulation abilities. nanoparticle tracking analysis (nta) was used to assess ev size and concentration while protein mass was measured via a bca assay. results: preliminary data suggests that priming mscs with 100 µm ethanol for 24 hours prior to ev collection results in a strong inhibition of il-6 production in stimulated murine macrophages. nta revealed that msc-ev yield increased by about two orders of magnitude in the bioreactor (1.40e12 ± 7.92e10) when compared with flasks (2.28e10 ± 2.81e9). protein measurements also indicated that ev production in the bioreactor (~7600 µg) was much greater compared with production in the flasks (~2400 µg). additionally, average protein content per ev was reduced in the bioreactor when compared with flask evs. regardless of tissue source. furthermore, comparison of adipose tissue-derived (ad) msc evs from three donors indicates varying pro-vascularization bioactivity between those donors evaluated in vitro via gap closure assay. similar results were observed for the bone marrow-derived (bm) msc ev donor groups. summary/conclusion: this work highlights the need for screening of donor derived-mscs before use for therapeutic ev production. additionally, standardized criteria for msc donor selection are needed before isolated msc evs can be used as a large-scale, repeatable therapeutic treatment. analysis of extracellular vesicle populations from malaria-infected erythrocytes by field-flow fractionation reveal distinct sub-sets alicia rojas a , paula abou-karam a , anna rivkin a , yael fridmann-sirkis b , yifat ofir-birin c and neta regev-rudzi c a department of biochemical sciences, weizmann institute of sciences, rehovot, israel, rehovot, israel; b wis, rehovot, israel; c weizmann institute of science, rehovot, israel introduction: malaria is one the most devastating infectious disease in the world and plasmodium falciparum (pf) represents the deadliest species. this parasite invades human red blood cells (rbcs) and releases extracellular vesicles (evs) carrying dna, rna and protein cargo components which are involved in the pathogenesis of the disease. recently, it has been shown in mammalian systems that evs are subdivided into different subpopulations, each with a distinct biological function. however, it is still unknown whether pfinfected rbcs (pf-evs) release different ev subpopulations with distinct cargo. methods: we isolated evs from pf-infected and uninfected rbcs, pf-evs or ui-evs, respectively, using differential centrifugation. the ev pellet was subjected to field flow fractionation (fff). the different subpopulations were collected, concentrated with size-exclusion filters and evaluated by nanoparticle tracking analysis. additionally, the presence of ev markers (sr1 and hsp90) were examined by western blot analysis. results: the fff analysis showed four particle subpopulations derived from the pf-evs and five in the ui-evs. the first three subpopulations were similar in their detection signals in both samples, but the fourth subpopulation was consistently higher in ui-evs than in pf-evs. moreover, hsp90 was detected in subpopulations 3 and 4 of both pf-evs and ui-evs, whereas sr1 only in subpopulation 3. isev2020 abstract book summary/conclusion: pf-ev and ui-ev have similar separation profiles and proteins markers in their subpopulations, consistent with the fact that both samples are derived from host rbcs. additional data regarding the dna and rna cargo, as well as microscopic observations of the pf-ev and ui-ev subpopulations is necessary. this will clarify how malaria parasites sort their components into evs and which fractions are associated to immune evasion and pathogenesis. we have established a small size laboratory production of the microalgae culture in order to harvest the extracellular vesicles (evs) for pharmaceutical and medical uses. in this work we report on globular particles in the isolates from media of microalgae of two types, that we recognize as evs. we observed changes in their production at different temperatures and conditions. methods: samples were fixed by various combinations of aldehyde fixatives and/or osmium tetroxide. they were dehydrated in a graded series of ethanol, hexamethyldisilazane, and air dried. they were au/pd coated for inspection with scanning electron microscopes (sem) crossbeam 550 fib-sem gemini ii (zeiss, germany) and jsm-6500 f field emission scanning electron microscope (jeol ltd., tokyo, japan). results: microalgae were incubated overnight at 22°c and 37°c in growth medium and in growth medium supplemented with detergent. the samples obtained from the microalgae culture contained particles that we recognized as extracellular vesicles, however, these particles do not correspond to characteristic shapes of membrane enclosed entities without internal structure. increased temperature and/or presence of surfactant (triton x-100 and sodium dodecyl sulphate) stimulated formation of evs of different shapes and sizes. the isolates of these samples were rich with evs. in the presence of surfactant, the cell-walls detached from the cell and collapsed upon dehydration. this was documented by sem. summary/conclusion: focused ion beam technique revealed complex internal structure of the algae. it seems from the shapes of the observed structures that the particles deposited on the surface of the microalgae do not derive from budding of the membrane surface, but are instead shed by the cells from the cell interior upon the rupture of the cell wall. 