Andrea Németh

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection

Circulating extracellular vesicles have emerged as potential new biomarkers in a wide variety of diseases. Despite the increasing interest, their isolation and purification from body fluids remains challenging. Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates. Using flow cytometry, we found that the majority of circulating particles within the size range of extracellular vesicles lacked common vesicular markers. We identified most of these particles as lipoproteins (predominantly low-density lipoprotein, LDL) which mimicked the characteristics of extracellular vesicles and also co-purified with them. Based on biophysical properties of LDL this finding was highly unexpected. Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates. Furthermore, transmission electron microscopy showed an association of LDL with isolated vesicles upon in vitro mixing. This is the first study to show co-purification and in vitro association of LDL with extracellular vesicles and its interference with vesicle analysis. Our data point to the importance of careful study design and data interpretation in studies using blood-derived extracellular vesicles with special focus on potentially co-purified LDL.

Improved Characterization of EV Preparations Based on Protein to Lipid Ratio and Lipid Properties

In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody) and ganglioside GM1 (cholera toxin subunit B). We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition), may prove useful for quality control of extracellular vesicle related basic and clinical studies.

Differential detergent sensitivity of extracellular vesicle subpopulations.

Extracellular vesicles (including exosomes, microvesicles and apoptotic bodies) are currently attracting rapidly increasing attention from various fields of biology due to their ability to carry complex information and act as autocrine, paracrine and even endocrine intercellular messengers. In the present study we investigated the sensitivity of size-based subpopulations of extracellular vesicles to different concentrations of detergents including sodium dodecyl sulphate, Triton X-100, Tween 20 and deoxycholate. We determined the required detergent concentration that lysed each of the vesicle subpopulations secreted by Jurkat, THP-1, MiaPaCa and U937 human cell lines. We characterized the vesicles by tunable resistive pulse sensing, flow cytometry and transmission electron microscopy. Microvesicles and apoptotic bodies were found to be more sensitive to detergent lysis than exosomes. Furthermore, we found evidence that sodium dodecyl sulphate and Triton X-100 were more effective in vesicle lysis at low concentrations than deoxycholate or Tween 20. Taken together, our data suggest that a combination of differential detergent lysis with tunable resistive pulse sensing or flow cytometry may prove useful for simple and fast differentiation between exosomes and other extracellular vesicle subpopulations as well as between vesicular and non-vesicular structures.

Extracellular vesicles in cardiovascular disease: are they Jedi or Sith?

In the recent past, extracellular vesicles have become recognized as important players in cell biology and biomedicine. Extracellular vesicles, including exosomes, microvesicles and apoptotic bodies, are phospholipid bilayer‐enclosed structures found to be secreted by most if not all cells. Extracellular vesicle secretion represents a universal and highly conserved active cellular function. Importantly, increasing evidence supports that extracellular vesicles may serve as biomarkers and therapeutic targets or tools in human diseases. Cardiovascular disease undoubtedly represents one of the most intensely studied and rapidly growing areas of the extracellular vesicle field. However, in different studies related to cardiovascular disease, extracellular vesicles have been shown to exert diverse and sometimes discordant biological effects. Therefore, it might seem a puzzle whether these vesicles are in fact beneficial or detrimental to cardiovascular health. In this review we provide a general introduction to extracellular vesicles and an overview of their biological roles in cardiovascular diseases. Furthermore, we aim to untangle the various reasons for the observed discrepancy in biological effects of extracellular vesicles in cardiovascular diseases. To this end, we provide several examples that demonstrate that the observed functional diversity is in fact due to inherent differences among various types of extracellular vesicles.

Critical role of extracellular vesicles in modulating the cellular effects of cytokines

Under physiological and pathological conditions, extracellular vesicles (EVs) are present in the extracellular compartment simultaneously with soluble mediators. We hypothesized that cytokine effects may be modulated by EVs, the recently recognized conveyors of intercellular messages. In order to test this hypothesis, human monocyte cells were incubated with CCRF acute lymphoblastic leukemia cell line-derived EVs with or without the addition of recombinant human TNF, and global gene expression changes were analyzed. EVs alone regulated the expression of numerous genes related to inflammation and signaling. In combination, the effects of EVs and TNF were additive, antagonistic, or independent. The differential effects of EVs and TNF or their simultaneous presence were also validated by Taqman assays and ELISA, and by testing different populations of purified EVs. In the case of the paramount chemokine IL-8, we were able to demonstrate a synergistic upregulation by purified EVs and TNF. Our data suggest that neglecting the modulating role of EVs on the effects of soluble mediators may skew experimental results. On the other hand, considering the combined effects of cytokines and EVs may prove therapeutically useful by targeting both compartments at the same time.

Oxidative and other posttranslational modifications in extracellular vesicle biology.

Extracellular vesicles including exosomes, microvesicles and apoptotic vesicles, are phospholipid bilayer surrounded structures secreted by cells universally, in an evolutionarily conserved fashion. Posttranslational modifications such as oxidation, citrullination, phosphorylation and glycosylation play diverse roles in extracellular vesicle biology. Posttranslational modifications orchestrate the biogenesis of extracellular vesicles. The signals extracellular vesicles transmit between cells also often function via modulating posttranslational modifications of target molecules, given that extracellular vesicles are carriers of several active enzymes catalysing posttranslational modifications. Posttranslational modifications of extracellular vesicles can also contribute to disease pathology by e.g. amplifying inflammation, generating neoepitopes or carrying neoepitopes themselves.

Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA

Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells. Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes. Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin. DNA release on the surface of exosomes was not affected any further by cellular activation or apoptosis induction. Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes.

Biliary complications following orthotopic liver transplantation. The Hungarian experience

INTRODUCTION The authors summarize the characteristics of biliary complications following liver transplantation in the Hungarian liver transplant program. Aims were to analyze the frequency and the types of biliary complications as well as their effect on the patient and graft survival. The authors observed the known risk factors in the Hungarian practice, and they also try to find unknown risk factors for biliary complications. They review the therapy of biliary complications. METHOD In the retrospective study, patients were divided into two groups, with and without biliary complication after liver transplantation. These two groups were compared with many factors, and with the survivals. The biliary complication group was divided into two parts: those who had an early and those with a late biliary complication. These two new groups were also compared with the controls. The results are summarized in tables and statistical figures. Categorical variables are evaluated by chi 2 -test, continuous ones are with Levine Test (for homogenicity of means), Student T test and Mann-Whitney U-test. Cumulative survivals are computed with Kaplan-Meier log rank analysis. RESULTS Biliary complication appeared in 25% of the patients. The most frequent complications were stenosis (18%), biliary leakage (9%), biliary necrosis (6%), and ischaemic type of biliary lesions (3%). The 5-year survival is worse when biliary complications were diagnosed (55%) than without such a complication (66%). In the biliary complication group the retransplantation rate was higher (15%). The most frequent treatments were interventional radiologic methods (69%), surgical methods (17%), and the ERCP. CONCLUSIONS The rate of biliary complications met the international reviews. Risk factors for biliary complications were cholangitis, hepatic artery thrombosis and stenosis, high rate of intraoperative blood transfusions, and acute rejection. Biliary complications frequently associated with the initial poor function of the transplanted graft. Early biliary complications have a negative impact on patient survival, while late complications influence a decreased quality of life. Biliary complications were treated mostly by interventional radiologic procedures.

The recurrence of hepatitis C virus after liver transplantation

A hazai majatultetesi programban magas a hepatitis C-virus (HCV) okozta vegstadiumu majbetegseg miatt vegzett majatultetesek aranya. Celkituzes: A szerzok dolgozatukban elemzik a C-hepatitis miatt majatultetesen atesett betegek adatait. Modszer: Az 1995 ota vegzett 295 primer majatultetes adatainak retrospektiv elemzese: donor- es recipiens-, valamint perioperativ es tulelesi adatok, szerumvirus-RNS-titer, percutan majbiopsziak szovettani eredmenyei. Eredmenyek: A mutet 111 betegnel tortent HCV-fertozes miatt, ez az elvegzett majatultetesek 37,6%-a. A vizsgalt 111 beteg kozul 22 beteg (20%) a posztoperativ idoszakban, a virus kiujulasanak eszlelese elott, egyeb okbol meghalt. A 89 beteg kozul 16 esetben (18%) a virus visszatereset meg nem eszleltek, 73 betegnel (82%) azonban a virus kiujulasa szovettanilag igazolhato volt. Negyven betegnel (56%) a C-virus okozta hepatitis kiujulasat egy even belul eszleltek, kozuluk 28 esetben (39%) 6 honapon belul, 12 esetben hat honapon tul, de 1 even belul (17%), es 32 betegnel (44%) egy even tul. A vegstadiumu C-cirrhosis miatt majatultetett betegek kumulativ 1, 3, 5 es 10 eves tulelese 73%, 67%, 56% es 49% volt. A HCV-negativ, majatultetett betegeknel ezek az ertekek 80%, 74%, 70% es 70%, a kulonbseg szignifikans. A majgraft kumulativ tulelese HCV-pozitiv betegeknel 72%, 66%, 56% es 49% volt, mig HCV-negativ betegeknel 76%, 72%, 68% es 68%, itt nem szignifikans a kulonbseg. Korai kiujulas eseten szignifikansan magasabb szerumvirus-RNS-titert mertek az elso 6 honapban majatultetes utan. A majatultetes utan 6 honappal vett protokollbiopszia korai kiujulas eseten magasabb Knodell-pontszamot eredmenyezett, mint kesoi kiujulaskor. A fibrosisindex eseteben ez forditva volt. A majatultetestol az elso antiviralis kezelesig eltelt ido 1995–2002 kozott atlagosan 20 honap volt, 2003 ota 8 honap. Kovetkeztetesek: Az idosebb donorokbol szarmazo, marginalis majgraftok magasabb vertranszfuzio-igeny mellett torteno beultetese elorevetiti a hamarabb bekovetkezo virusrekurrenciat. Ezt a tendenciat erositi a posztoperativ akut rejectio es az emiatt adott szteroid boluskezeles. A kombinalt antiviralis kezeles protokollja kulonbozik az altalanosan alkalmazottol: az un. „stopszabaly” nem ervenyes. Virusnegativva a betegek csak kevesebb mint 10%-a valik, melynek a fenntartott immunszuppresszio az oka. A majatultetes utan koran, akar fel even belul elkezdett antiviralis kezeles a beteg- es grafttulelest pozitivan befolyasolja, es feltehetoen csokkenti a HCV-reinfekcio miatti retranszplantaciok szamat. A masodik majatultetesnel akkor varhatok jo eredmenyek, ha idoben tortenik, a recipiens meg megfelelo fizikai allapota mellett. Ennek megiteleseben a MELD-score segit. Kulcsszavak: majatultetes, hepatitis C-virus, interferon, rekurrencia, retranszplantacio, tuleles The recurrence of hepatitis C virus after liver transplantation. The main indication of the Hungarian Liver Transplant Program is liver cirrhosis caused by hepatitis C. Aim: Authors present the results of liver transplantations performed due to HCV infection. Method: The data (donor-, recipient-, perioperative characteristics, survival, serum titer of C RNA, histology) of 111 HCV positive recipients were evaluated, that are 37.6% of the 295 patients, who were transplanted since 1995 till the closure of this report. Results: Twenty-two (22) of them (20%) died in the early postoperative period, for other reasons, before the recurrence of the HCV was detectable. Among the 89 HCV-positive patients the recurrence of the HCV is still not detected in 16 cases (18%), and there is a histology-proven recurrence in 73 cases (82%). In 40 cases (56%) the viral recurrence was proven within 1 year after OLT, while in 32 cases (44%) over 1 year. The cumulative 1, 3, 5, and 10 years patient survival is 73%, 67%, 56% and 49%, among HCV-positive patients and 80%, 74%, 70% and 70% among HCV-negatives. The difference is significant. The cumulative graft survival at the same time points is 72%, 66%, 56% and 49% among HCV-positives and 76%, 72%, 68% and 68% among HCV-negatives, which is a non-significant difference. The serum titer of HCV-RNA was significantly higher among those HCV-patients who had an early viral recur