Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

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

Role of microRNAs and Exosomes in Helicobacter pylori and Epstein-Barr Virus Associated Gastric Cancers

Emerging evidence suggests that chronic inflammation caused by pathogen infection is connected to the development of various types of cancer. It is estimated that up to 20% of all cancer deaths is linked to infections and inflammation. In gastric cancer, such triggers can be infection of the gastric epithelium by either Helicobacter pylori (H. pylori), a bacterium present in half of the world population; or by Epstein-Barr virus (EBV), a double-stranded DNA virus which has recently been associated with gastric cancer. Both agents can establish lifelong inflammation by evolving to escape immune surveillance and, under certain conditions, contribute to the development of gastric cancer. Non-coding RNAs, mainly microRNAs (miRNAs), influence the host innate and adaptive immune responses, though long non-coding RNAs and viral miRNAs also alter these processes. Reports suggest that chronic infection results in altered expression of host miRNAs. In turn, dysregulated miRNAs modulate the host inflammatory immune response, favoring bacterial survival and persistence within the gastric mucosa. Given the established roles of miRNAs in tumorigenesis and innate immunity, they may serve as an important link between H. pylori- and EBV-associated inflammation and carcinogenesis. Example of this is up-regulation of miR-155 in H. pylori and EBV infection. The tumor environment contains a variety of cells that need to communicate with each other. Extracellular vesicles, especially exosomes, allow these cells to deliver certain type of information to other cells promoting cancer growth and metastasis. Exosomes have been shown to deliver not only various types of genetic information, mainly miRNAs, but also cytotoxin-associated gene A (CagA), a major H. pylori virulence factor. In addition, a growing body of evidence demonstrates that exosomes contain genetic material of viruses and viral miRNAs and proteins such as EBV latent membrane protein 1 (LMP1) which are delivered into recipient cells. In this review, we focus on the dysregulated H. pylori- and EBV-associated miRNAs while trying to unveil possible causal mechanisms. Moreover, we discuss the role of exosomes as vehicles for miRNA delivery in H. pylori- and EBV-related carcinogenesis

Multiple Regulatory Roles of the Mouse Transmembrane Adaptor Protein NTAL in Gene Transcription and Mast Cell Physiology

<div><p>Non-T cell activation linker (NTAL; also called LAB or LAT2) is a transmembrane adaptor protein that is expressed in a subset of hematopoietic cells, including mast cells. There are conflicting reports on the role of NTAL in the high affinity immunoglobulin E receptor (FcεRI) signaling. Studies carried out on mast cells derived from mice with NTAL knock out (KO) and wild type mice suggested that NTAL is a negative regulator of FcεRI signaling, while experiments with RNAi-mediated NTAL knockdown (KD) in human mast cells and rat basophilic leukemia cells suggested its positive regulatory role. To determine whether different methodologies of NTAL ablation (KO vs KD) have different physiological consequences, we compared under well defined conditions FcεRI-mediated signaling events in mouse bone marrow-derived mast cells (BMMCs) with NTAL KO or KD. BMMCs with both NTAL KO and KD exhibited enhanced degranulation, calcium mobilization, chemotaxis, tyrosine phosphorylation of LAT and ERK, and depolymerization of filamentous actin. These data provide clear evidence that NTAL is a negative regulator of FcεRI activation events in murine BMMCs, independently of possible compensatory developmental alterations. To gain further insight into the role of NTAL in mast cells, we examined the transcriptome profiles of resting and antigen-activated NTAL KO, NTAL KD, and corresponding control BMMCs. Through this analysis we identified several genes that were differentially regulated in nonactivated and antigen-activated NTAL-deficient cells, when compared to the corresponding control cells. Some of the genes seem to be involved in regulation of cholesterol-dependent events in antigen-mediated chemotaxis. The combined data indicate multiple regulatory roles of NTAL in gene expression and mast cell physiology.</p></div

BMMCs with NTAL KD exhibit increased degranulation and calcium response.

<p>(A) IgE-sensitized BMMCs (WT, NTAL KO, NTAL KD, and WT pLKO) were stimulated for 30 minutes with various concentrations of Ag (TNP-BSA), and β-glucuronidase released into supernatant was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105539#s2" target="_blank">Materials and Methods</a>. Data represent means ± SE from 7–17 independent experiments performed in duplicates or triplicates). (B) IgE-sensitized BMMCs were stimulated with Ag [TNP-BSA at a concentration 100 ng/ml (Ag-100) or 500 ng/ml (Ag-500)], SCF (40 ng/ml), or both activators together. Data represent means ± SE from 6–20 independent experiments). (C–E) BMMCs were sensitized with IgE, loaded with Fura-2-AM (1 µg/ml), then stimulated with Ag (100 ng/ml TNP-BSA; C), SCF (40 ng/ml; D) or both activators together (E) and free intracellular Ca²⁺ was monitored by measuring fluorescence emission at 510 nm after excitation at 340 and 380 nm. Arrows indicate addition of Ag and/or SCF. Data are means ± SE from 11 (C), 7 (D) or 6 (E) independent experiments performed in duplicates. All data presented in A–E were obtained with BMMCs isolated from 3–5 mice. *^,+<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; in A, C and E, significant differences between NTAL KOs and WTs (asterisks) and NTAL KDs and WT pLKOs (crosslets) are shown.</p

BMMCs with NTAL KD exhibit enhanced actin depolymerization after stimulation with Ag or Ag + SCF.

<p>Cells were activated with Ag (250 ng/ml TNP-BSA; A), SCF (40 ng/ml; B) or Ag + SCF (C). At the indicated times, the cells were fixed, stained for F-actin with Alexa Fluor 488-phalloidin and analyzed by flow cytometry. Data were normalized to fluorescence of resting cells (similar in all cell types). Values indicate mean ± SE (n = 6). *^,+<i>p</i><0.05; **^,++<i>p</i><0.01; ***^,+++<i>p</i><0.001; significant differences between NTAL KOs and WTs (asterisks) and NTAL KDs vs WT pLKOs (crosslets) are shown.</p

A hypothetical model on the role of NTAL in mast cell activation and transcriptional regulation.

<p>(A) In nonactivated WT cells both adaptor proteins, LAT and NTAL, and FcεRI β and γ subunits only exhibit weak phosphorylation, low [Ca²⁺]_i and transcription corresponding to nonactivated cells (Transcription profile 1). (B) After Ag-mediated aggregation of the FcεRI-IgE complex, β and γ subunits of the FcεRI are tyrosine phosphorylated by LYN and SYK. SYK then phosphorylates NTAL and LAT and this leads to enhanced Ca²⁺ uptake and further propagation of the signal, including dramatic changes in transcriptional regulation (Transcription profile 2). (C) In nonactivated NTAL-deficient cells, LAT and FcεRI subunits are only weakly tyrosine phosphorylated and the cells exhibit slightly different transcriptional regulation when compared to WT cells (Transcription profile 3). (D) After FcεRI triggering of NTAL-deficient cells, β and γ subunits of the FcεRI are tyrosine phosphorylated as in B, but because of the absence of NTAL, LAT is more phosphorylated by SYK. This leads to enhanced mobilization of Ca²⁺ and other signaling events and transcriptional regulation which differs from the one in activated WT cells (Transcription profile 4).</p

Differences in transcriptional regulation between NTAL KO and WT cells.

<p>Differences in transcriptional regulation between NTAL KO and WT cells.</p

Decreased NTAL expression after shRNA silencing.

<p>(A) BMMCs were infected with five lentiviral shRNA constructs (NTAL KD 1–5) or empty pLKO.1 construct (WT pLKO). After selection in puromycin, the amount of NTAL was assessed by immunoblotting. For comparison, NTAL in noninfected WT and NTAL KO cells was also evaluated. Actin was used as a loading control. (B) Densitometry analysis of NTAL immunoblots. The data were normalized to the amount of NTAL in WT pLKO cells and that of actin. Means ± SD were calculated from 3–7 independent experiments. ***<i>p</i><0.001.</p

Principal component analysis of the microarrays.

<p>Each colored circle represents different cell type: NTAL KD (KD; red), NTAL KO (KO; blue), WT pLKO (pLKO; green), and WT (lilac). Each mouse from which the cells originated is identified by a colored numbers. The WT BMMCs isolated from mice 1–3 (blue numbers) were used not only as controls for NTAL KO cells, but also for obtaining NTAL KD and WT pLKO cells after lentiviral infection with NTAL shRNA or empty pLKO vector, respectively. The BMMC isolated from <i>Lat</i>^-/- mice 4–6 (lilac numbers) were used only as cells with NTAL KO. Treatment [Ag-activated cells (2h, pink) and nonactivated cells (0h, green)] is distinguished by ellipsoids. The arrays cluster according to the treatment groups showing separation along PC #1 and according to the type of cells showing separation along PC #2. The percentage values indicate the proportion of total variance described by each PC; PC #1 (X-axis), PC #2 (Y-axis), and PC #3 (Z-axis).</p

Primers used in quantitative RT-PCR.

<p>Primers used in quantitative RT-PCR.</p