The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Background & Aims Microparticles released into the bloodstream upon activation or apoptosis of CD4+ and CD8+ T cells correlate with inflammation, determined by histologic analysis, in patients with chronic hepatitis C (CHC). Patients with nonalcoholic fatter liver (NAFL) or nonalcoholic steatohepatitis (NASH) can be differentiated from those with CHC based on activation of distinct sets of immune cells in the liver. Methods We compared profiles of circulating microparticles from patients with NAFL and NASH (n=67) to those with CHC (n=42), compared with healthy individuals (controls) using flow cytometry; the profiles were correlated with inflammation grade and fibrosis stage, based on histologic analyses. We assessed the ability of the profiles determine the severity of inflammation and fibrosis, based on serologic and histologic analyses. Results Patients with CHC had increased levels of microparticles from CD4+ and CD8+ T cells; the levels correlated with disease severity, based on histologic analysis and levels of alanine aminotransferase (ALT). Patients with NAFL or NASH had significant increases in numbers of microparticles from invariant natural killer T (iNKT) cells and macrophages/monocytes (CD14+), which mediate pathogenesis of NASH. Microparticles from CD14+ and iNKT cells correlated with levels of ALT and severity of NASH (based on histology). Levels of microparticles could differentiate between patients with NAFL or NASH and those with CHC, or either group of patients and controls (area under the receiver operating characteristic curves ranging from 0.56 to 0.99). Conclusions Quantification of immune cell microparticles from serum samples can be used to assess the extent and characteristics of hepatic inflammation in patients with chronic liver disease.

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Radiofrequency ablation (RFA) is a potentially curative therapy for hepatocellular carcinoma (HCC). However, incomplete RFA can induce accelerated invasive growth at the periphery. The mechanisms underlying the RFA-induced tumor promotion remain largely unexplored. Three human HCC cell lines were exposed to 45 C-55 C for 10 minutes, simulating the marginal zone of RFA treatment. At 5-12 days posttreatment cell proliferation, parameters of epithelial-mesenchymal transition (EMT), and activation of mitogen-activated protein kinases were analyzed. Livers from patients with viral hepatitis without and with HCC (n 5 114) were examined to confirm the relevance of altered kinase patterns. In vivo tumorigenic potential of heat-treated versus untreated HCC cells was studied in nude mice. Heating to 55 C killed all HCC cells, whereas 65%-85% of cells survived 48 C-50 C, developing spindle-like morphology and expressing CD133, cytokeratin (CK)7, CK19, procollagen-a1(I), and Snail at day 5 after heat exposure, which returned to baseline at day 12. Heat-exposed HCC cells showed enhanced proliferation and prominent activation of p46-Shc (Src homology and collagen) and downstream extracellular signalrelated kinase (Erk)1/2. In patients, Shc expression correlated with malignant potential and overall survival. Blocking Erk1/2 reduced proliferation and EMT-like changes of heat-treated HCC cells. Implantation of heat-exposed HEPG2 cells into nude mice induced significantly larger, more aggressive tumors than untreated cells. Conclusions: Sublethal heat treatment skews HCC cells toward EMT and transforms them to a progenitor-like, highly proliferative cellular phenotype in vitro and in vivo, which is driven significantly by p46Shc-Erk1/2. Suboptimal RFA accelerates HCC growth and spread by transiently inducing an EMT-like, more aggressive cellular phenotype. (HEPATOLOGY 2013;58:1667-1680 R adiofrequency ablation (RFA) is accepted as a potentially curative therapy for the early stages of primary hepatocellular carcinoma (HCC). 1 RFA induces tumor necrosis with low complication rates and is superior to percutaneous ethanol injection in tumor ablation.2 However, suboptimal RFA treatment for HCC has been reported as a risk factor of early diffuse recurrence.3 Large tumor size is a major risk factor of Abbreviations: 7-AAD, 7-aminoactinomycin D; AFP, alpha-fetaprotein; ALB, albumin; BSA, bovine serum albumin; CFSE, carboxyfluorescein succinimidyl ester; CHC, chronic hepatitis C; CK, cytokeratin; COL1A1, type I procollagen; Ct, cycle threshold; DI, division indices; DMEM, Dulbecco's modified Eagle's medium; EGFR, epidermal growth factor receptor; EMT, epithelial-mesenchymal transition; Erk, extracellular signal-related kinase; ETW, estimated tumor weight; FACS, fluorescein-activated cell sorting; FBS, fetal bovine serum; FCM, flow cytometry; FGFR, fibroblast growth factor receptor; HCC, hepatocellular carcinoma; HSP, heat shock protein; IGF-1R, insulin-like growth factor 1 receptor; JNK, c-Jun N-terminal kinase; LI, labeling indices; MAP...

Microparticles (MP) are small cell membrane vesicles which are released from cells during apoptosis or activation. While circulating platelet MP have been studied in some detail, the existence and functional role of T cell MP remain elusive. We show that blood from patients with active hepatitis C (ALT>100 IU/ml) contains elevated numbers of T cell MP compared to patients with mild hepatitis C (ALT<40 IU/ml) and healthy controls. T cell MP fuse with cell membranes of hepatic stellate cells (HSC), the major effector cells for excess matrix deposition in liver fibrosis and cirrhosis. MP uptake is partly ICAM-1 dependent and leads to activation of NFkB and ERK1/2 and subsequent upregulation of fibrolytic genes in HSC, to downregulation of procollagen α1(I) mRNA, and blunting of profibrogenic activities of TGFβ1. Ex vivo the induced fibrolytic activity is evident in MP derived from activated CD4+ T cells, and highest with MP from activated and apoptotic CD8+ T cells. Mass spectrometry, FACS analysis and function blocking antibodies revealed CD147/Emmprin as candidate transmembrane molecule in HSC fibrolytic activation by CD8+ T cell MP. We conclude that 1) circulating T cell MP are a novel diagnostic marker for inflammatory liver diseases, and 2) in vivo induction of T cell MP may be a novel strategy to induce regression of liver fibrosis.

The terms microparticles (MPs) and microvesicles (MVs) refer to large extracellular vesicles (EVs) generated from a broad spectrum of cells upon its activation or death by apoptosis. The unique surface antigens of MPs/MVs allow for the identification of their cellular origin as well as its functional characterization. Two basic aspects of MP/MV functions in physiology and pathological conditions are widely considered. Firstly, it has become evident that large EVs have strong procoagulant properties. Secondly, experimental and clinical studies have shown that MPs/MVs play a crucial role in the pathophysiology of inflammation-associated disorders. A cardinal feature of these disorders is an enhanced generation of platelets-, endothelial-, and leukocyte-derived EVs. Nevertheless, anti-inflammatory effects of miscellaneous EV types have also been described, which provided important new insights into the large EV-inflammation axis. Advances in understanding the biology of MPs/MVs have led to the preparation of this review article aimed at discussing the association between large EVs and inflammation, depending on their cellular origin.

The high mortality rate of cholangiocarcinoma (CCA) is due, in part, to the lack of non‐invasive approaches able to accurately detect this silent tumour at early stages, when therapeutic options can be potentially curative or may at least increase the overall survival of patients. The fact that the majority of CCA tumours are not linked to any known aetiological factor highly compromises the monitoring of patients at risk for tumour development and also their early diagnosis. Combination of clinical/biochemical features, imaging techniques and analysis of non‐specific tumour biomarkers in serum are commonly used to help in the diagnosis of CCA, but tumour biopsy is usually required to confirm the diagnosis. Moreover, no prognostic biomarkers are currently used in the clinical setting, deserving more innovative research, and international validation and consensus. Important efforts have been made in the last few years to identify accurate non‐invasive biomarkers, by using innovative techniques and high‐throughput omics technologies. This review summarizes and discusses the advances in the investigation of novel diagnostic and prognostic biomarkers in CCA and envisions the future directions in this field of research.

Soluble Ags devoid of inflammatory stimuli, derived for example from self-serum or food proteins, induce T cell tolerance, predominantly in the spleen. In this study, we describe an additional role of the kidney-renal LN (rLN) system in tolerogenic presentation of circulating soluble Ags. Protein below albumin molecular mass constitutively passed the kidney glomerular filter and was concentrated in the tubular compartment. Enriched filterable Ag was endocytosed by kidney dendritic cells (kDCs). Simultaneously, it was transported cell independently within 2 min to DCs resident in rLNs. These DC phenotypically differed from kDCs carrying filterable Ag, and used a distinct mechanism, mannose receptor-mediated endocytosis, to internalize Ag. They activated specific CD8+ T cells, which subsequently proliferated without producing effector cytokines or developing cytotoxic activity, showed a curtailed lifespan and signs of apoptosis. Such T cell tolerization was independent of steady-state migratory kDC, because it occurred also when nephrectomy was performed soon after Ag injection. These findings demonstrate that the kidney dispatches concentrated blood-borne Ags to the rLNs, where they are captured by resident DCs, resulting in CD8+ T cell cross-tolerance. This mechanism may contribute to avoiding immunity against innocuous circulating protein Ags below albumin size.