A Coxsackievirus B1-mediated nonlytic Extracellular Vesicle-to-cell mechanism of virus transmission and its possible control through modulation of EV release.

Like most non-enveloped viruses, CVB1 mainly uses cell lysis to spread. Details of a nonlytic virus transmission remain unclear. Extracellular Vesicles (EVs) transfer biomolecules between cells. We show that CVB1 entry into HeLa cells results in apoptosis and release of CVB1-induced 'medium-sized' EVs (CVB1i-mEVs). These mEVs (100-300 nm) harbour CVB1 as shown by immunoblotting with anti-CVB1-antibody; viral capsids were detected by transmission electron microscopy and RT-PCR revealed CVB1 RNA. The percentage of mEVs released from CVB1-infected HeLa cells harbouring virus was estimated from TEM at 34 %. Inhibition of CVB1i-mEV production, with calpeptin or siRNA knockdown of CAPNS1 in HeLa cells limited spread of CVB1 suggesting these vesicles disseminate CVB1 virions to new host cells by a nonlytic EV-to-cell mechanism. This was confirmed by detecting CVB1 virions inside HeLa cells after co-culture with CVB1i-mEVs; EV release may also prevent apoptosis of infected cells whilst spreading apoptosis to secondary sites of infection.

Extremely low-frequency magnetic fields significantly enhance the cytotoxicity of methotrexate and can reduce migration of cancer cell lines via transiently induced plasma membrane damage.

Extra Low-frequency Magnetic Fields (ELF-MFs) significantly enhance cellular uptake of methotrexate by inducing transient plasma membrane pores/damage. This enhanced 'dose loading' of methotrexate via the electromagnetically induced membrane pores leads to similar outcomes as the normal control while using significantly smaller therapeutic doses in vitro when compared to non-ELF-MF treated control. Approximately 10% of the typical therapeutic dose yielded similar results when used with ELF-MF. ELF-MFs increase PC12, THP-1 and HeLa proliferation in vitro (120% of the control). Analysis of adherent cells demonstrate significantly less migration towards an induced scratch injury (20 µm in 24 h when compared to a control). Our results suggest an important role for the use of ELF-MFs in the treatment of tumours that opens some new and exciting possibilities including using smaller therapeutic doses of chemotherapeutic agents and disrupting tumour metastasis.

Sigma-1 receptors modulate neonatal Nav1.5 ion channels in breast cancer cell lines.

The main aim of this study was to investigate a possible functional connection between sigma-1 receptors and voltage-gated sodium channels (VGSCs) in human breast cancer cells. The hypothesis was that sigma-1 drugs could alter the metastatic properties of breast cancer cells via the VGSC. Evidence was found for expression of sigma-1 receptor and neonatal Nav1.5 (nNav1.5) expression in both MDA-MB-231 and MDA-MB-468 cells. Sigma-1 drugs (SKF10047 and dimethyltryptamine) did not affect cell proliferation or migration but significantly reduced adhesion to the substrate. Silencing sigma-1 receptor expression by siRNA similarly reduced the adhesion. Blocking nNav1.5 activity with a polyclonal antibody (NESOpAb) targeting an extracellular region of nNav1.5 also reduced the adhesion in both cell lines. Importantly, the results of combined treatments with NESOpAb and a sigma-1 drug or sigma-1 siRNA suggested that both treatments targeted the same mechanism. The possibility was tested, therefore, that the sigma-1 receptor and the nNav1.5 channel formed a physical, functional complex. This suggestion was supported by the results of co-immunoprecipitation experiments. Furthermore, application of sigma-1 drugs to the cells reduced the surface expression of nNav1.5 protein, which could explain how sigma-1 receptor activation could alter the metastatic behaviour of breast cancer cells. Overall, these results are consistent with the idea of a sigma-1 protein behaving like either a "chaperone" or a regulatory subunit associated with nNav1.5.

Prostate cancer cells stimulated by calcium-mediated activation of protein kinase C undergo a refractory period before re-releasing calcium-bearing microvesicles.

MVs are released in response to several stress agents, in an attempt to prevent continued cellular damage. After an initial stimulus of prostate cancer cells with sublytic C5b-9 and activation of MV release through PKC, cells take at least 20 min to fully recover their ability to microvesiculate. This release of MVs through activation of sublytic C5b-9 was inhibited by the PKC inhibitor bisindoylmaleimide I but not the Rho kinase inhibitor, Y27632. After stimulus there is a rise of 79 nMs(-1) over 11 s, reaching a peak [Ca(2+)]i of 920 nM. The concentration of cytosolic calcium then falls steadily at 2.4 nMs(-1) over 109 s reaching baseline levels (50-100 nM) within 10-15 min. In PC3 cells the rate of release of MVs from stimulated cells also reaches a minimum within 10-15 min. Using fura-2 AM-loaded cells, upon stimulation, cells were found to release MVs with a concentration of intravesicular calcium estimated at ∼ 430 nM.

Microvesicles released constitutively from prostate cancer cells differ biochemically and functionally to stimulated microvesicles released through sublytic C5b-9.

We have classified microvesicles into two subtypes: larger MVs released upon stimulation of prostate cancer cells, sMVs, and smaller cMVs, released constitutively. cMVs are released as part of cell metabolism and sMVs, released at 10-fold higher levels, produced upon activation, including sublytic C5b-9. From electron microscopy, nanosight tracking analysis, dynamic light scattering and flow cytometry, cMVs (194-210 nm in diameter) are smaller than sMVs (333-385 nm). Furthermore, using a Quartz Crystal Microbalance measuring changes in resonant frequency (Δf) that equate to mass deposited on a sensor, an sMV and a cMV are estimated at 0.267 and 0.241 pg, respectively. sMVs carry more calcium and protein, express higher levels of lipid rafts, GPI-anchored CD55 and phosphatidylserine including deposited C5b-9 compared to cMVs. This may allude to biological differences such as increased bound C4BP on sMVs inhibiting complement more effectively.

Blood/plasma secretome and microvesicles.

A major but hitherto overseen component of the blood/plasma secretome is that of extracellular vesicles (EVs) which are shed from all blood cell types. These EVs are made up of microvesicles (MVs) and exosomes. MVs, 100nm-1µm in diameter, are released from the cell surface, and are a rich source of non-conventionally secreted proteins lacking a conventional signal peptide, and thus not secreted by the classical secretory pathways. Exosomes are smaller vesicles (≤100nm) having an endocytic origin and released upon multivesicular body fusion with the plasma membrane. Both vesicle types play major roles in intercellular cross talk and constitute an important component of the secretome especially in the area of biomarkers for cancer. The release of EVs, which are found in all the bodily fluids, is enhanced in cancer and a major focus of cancer proteomics is therefore targeted at EVs. The blood/plasma secretome is also a source of EVs, potentially diagnostic of infectious disease, whether from EVs released from infected cells or from the pathogens themselves. Despite the great excitement in this field, as is stated here and in other parts of this Special issue entitled: An Updated Secretome, much of the EV research, whether proteomic or functional in nature, urgently needs standardisation both in terms of nomenclature and isolation protocols. This article is part of a Special Issue entitled: An Updated Secretome.

Label-free real-time acoustic sensing of microvesicle release from prostate cancer (PC3) cells using a Quartz Crystal Microbalance.

Using a Quartz Crystal Microbalance with dissipation monitoring, QCM-D (label-free system) measuring changes in resonant frequency (Δf) that equate to mass deposited on a sensor, we showed the attachment, over a 60min period, of a monolayer of PC3 cells to the gold electrodes of the quartz crystal sensor, which had been rendered hydrophilic. That MVs were released upon BzATP stimulation of cells was confirmed by NTA analysis (average 250nm diameter), flow cytometry, showing high phosphatidylserine exposition and by fluorescent (Annexin V Alexa Fluor® 488-positive) and electron microscopy. Over a period of 1000s (16.7min) during which early apoptosis increased from 4% plateauing at 10% and late apoptosis rose to 2%, the Δf increased 20Hz, thereupon remaining constant for the last 1000s of the experiment. Using the Sauerbrey equation, the loss in mass, which corresponded to the release of 2.36×10(6)MVs, was calculated to be 23ng. We therefore estimated the mass of an MV to be 0.24pg. With the deposition on the QCM-D of 3.5×10(7)MVs over 200s, the decrease in Δf (Hz) gave an estimate of 0.235pg per MV.

Pulsed extremely low-frequency magnetic fields stimulate microvesicle release from human monocytic leukaemia cells.

Microvesicles are released from cell surfaces constitutively during early apoptosis or upon activation with various stimuli including sublytic membrane attack complex (MAC). This study shows that an alternating current, pulsed, extremely low-frequency electromagnetic field (0.3 µT at 10 Hz, 6V AC) induced transient plasma membrane damage that allowed calcium influx. This in turn caused a release of stimulated microvesicles (sMV). When extracellular calcium was chelated with EGTA, sMV biogenesis initiated by ELFMF was markedly reduced and the reduction was less than when the stimulation was the deposition of sublytic MAC. This suggested that pulsed ELFMF resulted in transcellular membrane pores causing organelles to leak additional calcium into the cytoplasm (which EGTA would not chelate) which itself can lead to sMV release.

Isolation and characterization of extracellular vesicles and future directions in diagnosis and therapy.

Extracellular vesicles (EVs) are a unique and heterogeneous class of lipid bilayer nanoparticles secreted by most cells. EVs are regarded as important mediators of intercellular communication in both prokaryotic and eukaryotic cells due to their ability to transfer proteins, lipids and nucleic acids to recipient cells. In addition to their physiological role, EVs are recognized as modulators in pathological processes such as cancer, infectious diseases, and neurodegenerative disorders, providing new potential targets for diagnosis and therapeutic intervention. For a complete understanding of EVs as a universal cellular biological system and its translational applications, optimal techniques for their isolation and characterization are required. Here, we review recent progress in those techniques, from isolation methods to characterization techniques. With interest in therapeutic applications of EVs growing, we address fundamental points of EV-related cell biology, such as cellular uptake mechanisms and their biodistribution in tissues as well as challenges to their application as drug carriers or biomarkers for less invasive diagnosis or as immunogens. This article is categorized under: Diagnostic Tools > Biosensing Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease.

Human plasma membrane-derived vesicles halt proliferation and induce differentiation of THP-1 acute monocytic leukemia cells.

Plasma membrane-derived vesicles (PMVs) are small intact vesicles released from the cell surface that play a role in intercellular communication. We have examined the role of PMVs in the terminal differentiation of monocytes. The myeloid-differentiating agents all-trans retinoic acid/PMA and histamine, the inflammatory mediator that inhibits promonocyte proliferation, induced an intracellular Ca(2+)-mediated PMV (as opposed to exosome) release from THP-1 promonocytes. These PMVs cause THP-1 cells to enter G(0)-G(1) cell cycle arrest and induce terminal monocyte-to-macrophage differentiation. Use of the TGF-ß receptor antagonist SB-431542 and anti-TGF-ß1 Ab showed that this was due to TGF-ß1 carried on PMVs. Although TGF-ß1 levels have been shown to increase in cell culture supernatants during macrophage differentiation and dendritic cell maturation, the presence of TGF-ß1 in PMVs is yet to be reported. In this study, to our knowledge we show for the first time that TGF-ß1 is carried on the surface of PMVs, and we confirm the presence within PMVs of certain leaderless proteins, with reported roles in myeloid cell differentiation. Our in vitro findings support a model in which TGF-ß1-bearing PMVs, released from promonocytic leukemia cells (THP-1) or primary peripheral blood monocytes on exposure to sublytic complement or after treatment with a differentiation therapy agent, such as all-trans retinoic acid, significantly reduce proliferation of THP-1 cells. Such PMVs also induce the terminal differentiation of primary peripheral blood monocytes as well as THP-1 monocytes.

Human plasma membrane-derived vesicles inhibit the phagocytosis of apoptotic cells--possible role in SLE.

Plasma membrane-derived vesicles (PMVs) also known as microparticles, are small membrane-bound vesicles released from the cell membrane via blebbing and shedding. PMVs have been linked with various physiological functions as well as pathological conditions such as inflammation, autoimmune disease and cardiovascular disease. PMVs are characterised by the expression of phosphatidylserine (PS) on the plasma membrane. PS, also expressed on apoptotic cells (ACs) enables macrophages to phagocytose ACs. As it is widely known that PMV production is increased during apoptosis, we were able to show that PMVs could compete dose dependently with ACs for the PS receptor on macrophages, so reducing phagocytosis of ACs. In a clinical setting this may result in secondary necrosis and further pathological conditions. In SLE in which there are raised PMV levels, there is an anti-phospholipid-mediated increase in PMV release, which can be abrogated by depletion of IgG. Our work provides an insight into how PMVs may play a role in the aetiology of autoimmune disease, in particular SLE.

Plasma mEV levels in Ghanain malaria patients with low parasitaemia are higher than those of healthy controls, raising the potential for parasite markers in mEVs as diagnostic targets.

This study sought to measure medium-sized extracellular vesicles (mEVs) in plasma, when patients have low Plasmodium falciparum early in infection. We aimed to define the relationship between plasma mEVs and: (i) parasitaemia, (ii) period from onset of malaria symptoms until seeking medical care (patient delay, PD), (iii) age and (iv) gender. In this cross-sectional study, n = 434 patients were analysed and Nanosight Tracking Analysis (NTA) used to quantify mEVs (vesicles of 150-500 nm diameter, isolated at 15,000 × g, ß-tubulin-positive and staining for annexin V, but weak or negative for CD81). Overall plasma mEV levels (1.69 × 1010 mEVs mL-1) were 2.3-fold higher than for uninfected controls (0.51 × 1010 mEVs mL-1). Divided into four age groups, we found a bimodal distribution with 2.5- and 2.1-fold higher mEVs in infected children (<11 years old [yo]) (median:2.11 × 1010 mEVs mL-1) and the elderly (>45 yo) (median:1.92 × 1010 mEVs mL-1), respectively, compared to uninfected controls; parasite density varied similarly with age groups. There was a positive association between mEVs and parasite density (r = 0.587, p < 0.0001) and mEVs were strongly associated with PD (r = 0.919, p < 0.0001), but gender had no effect on plasma mEV levels (p = 0.667). Parasite density was also exponentially related to patient delay. Gender (p = 0.667) had no effect on plasma mEV levels. During periods of low parasitaemia (PD = 72h), mEVs were 0.93-fold greater than in uninfected controls. As 75% (49/65) of patients had low parasitaemia levels (20-500 parasites µL-1), close to the detection limits of microscopy of Giemsa-stained thick blood films (5-150 parasites µL-1), mEV quantification by NTA could potentially have early diagnostic value, and raises the potential of Pf markers in mEVs as early diagnostic targets.

Effects of Acute and Chronic Exercise on Immunological Parameters in the Elderly Aged: Can Physical Activity Counteract the Effects of Aging?

Immunosenescence is characterized by deterioration of the immune system caused by aging which induces changes to innate and adaptive immunity. Immunosenescence affects function and phenotype of immune cells, such as expression and function of receptors for immune cells which contributes to loss of immune function (chemotaxis, intracellular killing). Moreover, these alterations decrease the response to pathogens, which leads to several age-related diseases including cardiovascular disease, Alzheimer's disease, and diabetes in older individuals. Furthermore, increased risk of autoimmune disease and chronic infection is increased with an aging immune system, which is characterized by a pro-inflammatory environment, ultimately leading to accelerated biological aging. During the last century, sedentarism rose dramatically, with a concomitant increase in certain type of cancers (such as breast cancer, colon, or prostate cancer), and autoimmune disease. Numerous studies on physical activity and immunity, with focus on special populations (i.e., people with diabetes, HIV patients) demonstrate that chronic exercise enhances immunity. However, the majority of previous work has focused on either a pathological population or healthy young adults whilst research in elderly populations is scarce. Research conducted to date has primarily focused on aerobic and resistance exercise training and its effect on immunity. This review focuses on the potential for exercise training to affect the aging immune system. The concept is that some lifestyle strategies such as high-intensity exercise training may prevent disease through the attenuation of immunosenescence. In this context, we take a top-down approach and review the effect of exercise and training on immunological parameters in elderly at rest and during exercise in humans, and how they respond to different modes of training. We highlight the impact of these different exercise modes on immunological parameters, such as cytokine and lymphocyte concentration in elderly individuals.

Inhibition of microvesiculation sensitizes prostate cancer cells to chemotherapy and reduces docetaxel dose required to limit tumor growth in vivo.

Microvesicles shed from cells carry constituents of the cell cytoplasm, including, of importance in multidrug resistance to cancer chemotherapy, drugs that the tumor cell attempts to efflux. To see whether such drugs could be used at lower concentrations with the same efficacy, it was first shown that microvesiculation of prostate cancer (PCa) cells, PC3, could be inhibited pharmacologically with calpeptin (calpain inhibitor) and by siRNA (CAPNS1). In cells treated with docetaxel (DTX), this inhibition resulted in a third-fold increase in intracellular concentrations of DTX. As a result, 20-fold lower concentrations of DTX (5 nM) could be used, in the presence of calpeptin (20 µM) inducing the same degree of apoptosis after 48 h in PC3 cells, as 100 nM of DTX alone. Inhibition of microvesiculation similarly improved combination chemotherapy (DTX and methotrexate). In a mouse xenograft model of PCa, DTX (0.1 mg/kg) together with calpeptin (10 mg/kg), administered i.p., significantly reduced tumor volumes compared to DTX alone (0.1 mg/kg) and brought about the same reductions in tumor growth as 10 mg/kg of DTX alone. As well as further reducing vascularization, it also increased apoptosis and reduced proliferation of PC3 cells in tumor xenografts.

Microvesicles in health and disease.

Microvesicles (or MVs) are plasma membrane-derived vesicles released from most eukaryotic cells constitutively during early apoptosis or at higher levels after chemical or physical stress conditions. This review looks at some of the functions of MVs in terms of intercellular communication and ensuant signal transduction, including the transport of proteins (unconventional protein export) as well as of mRNA and microRNA. MVs also have roles in membrane repair, the removal of misfolded proteins, and in the control of apoptosis. We also discuss the role MVs have been shown to have in invasive growth and metastasis as well as in hypoxia in tumours and cerebral ischaemia. The association of MVs in infectious and autoimmune disease is also summarised together with their possible use as therapeutic agents.

A filtration-based protocol to isolate human plasma membrane-derived vesicles and exosomes from blood plasma.

The methods of Plasma Membrane-derived Vesicle (PMV) isolation and quantification vary considerably in the literature and a new standard needs to be defined. This study describes a novel filtration method to isolate PMVs in plasma, which avoids high speed centrifugation, and to quantify them using a Becton Dickinson (BD) FACS Calibur™ flow cytometer, as annexin V-positive vesicles, larger than 0.2 µm in diameter. Essentially microvesicles (which comprise a mixture of PMVs and exosomes) from citrate plasma were sonicated to break up clumped exosomes, and filtered using Millipore 0.1 µm pore size Hydrophilic Durapore membranes in Swinnex 13 mm filter holders. Phosphatidylserine-positive PMVs detected with annexin V-PE were quantified using combined labelling and gating strategies in conjunction with Polysciences Polybead Microspheres (0.2 µm) and BDTrucount tubes. The PMV absolute count was calculated on the analysis template using the Trucount tube lot number information and expressed in PMV count/ml. Having estimated a normal reference range (0.51×10(5)-2.82×10(5) PMVs/ml) from a small sample of human donors, using the developed method, the effect of certain variables was investigated. Variations such as freezing of samples and gender status did not significantly alter the PMV absolute count, and with age plasma PMV levels were only marginally reduced. Smokers appeared to have reduced PMV levels. Nicotine, as for calpeptin was shown to dose-dependently (from 10 up to 50 µM) reduce levels of early apoptosis in THP-1 monocytes and to decrease the level of PMV release. Fasting individuals had 2-3 fold higher PMV absolute counts compared to non-fasting subjects.