Coxsackie virus entry and spread in HeLa cells is aided by microvesicle release

Microvesicles(MVs) released from plasma membrane expressing surface phosphatidylserine and ranging from 0.2-≤1 m in diameter are reported to carry various membrane proteins, lipids and cytoplasmic components characteristic of the parental cell (1). Coxsackievirus B (CVB), a member of the enterovirus family is the main cause of meningitis and encephalitis in infants which may result in neurodevelopmental defects. Calpains are calcium-dependant cysteine proteases that degrade cytoplasmic and cytoskeletal proteins. They regulate a variety of actin-dependant cellular processes such as microvesiculation. CVB1 requires calpain activation for both entry and virus replication. Here, we show that knocking down calpain, using approaches such as small interfering RNA (siRNA), culminates in reduction of MV release, as we showed before with another intracellular pathogen, the protozoan parasite, Trypanosoma cruzi (2). The reduction in MV release then abrogates CVB1 entry and spread in HeLa cells. The calpain inhibitor calpeptin also caused similar reduction in CVB1 entry and spread to healthy target cells. Together, our findings provide evidence that CVB1 infected HeLa cells enhance MV production, and these MVs aid the spread of infection. Furthermore, inhibition of MV release using siRNA results in inhibition of CVB1 entry and spread.Peer reviewedFinal Accepted Versio

Cancer cell expulsion of anticancer drugs through shedding of microvesicles: association with drug resistance and tumour survival

Microvesicles (MVs) are small (0.1-≤1 µm in diameter) heterogeneous vesicles released from cells constitutively or upon activation, that mediate intercellular communication. Multi-drug resistance (MDR) has been defined as the ability of cancer cells to survive after treatment with various drugs. However, the mechanism(s) used by cancer cells to evade apoptosis induced by anticancer drugs remain unclear and was the subject of our investigation. Here we report a novel mechanism of cancer cell expulsion of anticancer drugs through the release of MVs, followed by the recruitment of lysosomes to the site of release to repair the resulting damage. In addition, we show for the first time that inhibition of MV release by pretreatment of PC3M cells with the calpain inhibitor, calpeptin, sensitizes cancer cells to drug-elicited apoptosis mediated by the addition of methotrexate (MTX) and docetoxel (DOC) using at least 10-fold lower concentrations, both in vitro and in vivo. Treatment of cancer patients with MET or DOC leads to significant side effects due to the use of higher doses. Here we show that these drugs when administered together with calpeptin can be given at doses 100 times lower and still induce effective killing of target cancer cells. Overall our studies shed light on the role of MV release in cancer cell expulsion of anticancer drugs and subsequent evasion and survival from apoptosis and suggest new combination therapies for existing cancer drugs.Peer reviewedFinal Accepted Versio

Characterisation of microvesicles released from cells constitutively and upon stimulation

Constitutively released microvesicles (cMVs) are released as a part of normal cell physiology and this is summarised in Fig. 1 (1). However stimulated microvesicles (sMVs) are released as a result of a number of possible stress factors (2, 3). We found sMVs to be released in higher numbers than cMVs, typically ten-fold higher numbers, in the same time frame, and where the stress factor was a pharmacological agent, the microvesiculation was an attempt to release this chemical stress factor. Using a mass sensing technique, the sMVs were released over a 15 min period after stimulation. Using sizing beads on a flow cytometer and by transmission electron microscopy the cMVs were typically smaller (less than 300 nm in diameter) than sMVs (300-500 nm in diameter). However cMVs were found to carry more protein. By contrast, phosphatidylserine expression was greater on the larger sMVs, which also more effectively inhibited complement-mediated lysis.Peer reviewedFinal Accepted Versio

Human skeletal muscle derived microvesicles induce apoptosis in highly metastatic prostate cancer cells

The human skeletal muscle is a highly vascularised tissue that contributes approximately 40% of the total body mass. These two factors make it a prime target for secondary cancer metastases. Surprisingly, malignant cancers rarely metastasize to the skeletal muscle. Only 1.6% of all soft tissue sarcomas (muscle) examined are metastatic in origin. Various researchers over the years have therefore tried to elucidate the cellular and molecular mechanisms contributing to this rarity of secondary metastasis but they still remain obscure. Although some have postulated high levels of lactic acid or reported factors such as adenosine released by the skeletal muscle cell to create a toxic environment for secondary tumour development, the role of skeletal muscle microvesicles (MVs) on tumour cells is yet to be reported. In previous work we showed MVs capable of fusing with target cells. In this study, we show that MVs derived from human skeletal muscle cells (HSkMC) have a cytotoxic effect (inducing 30% apoptosis) upon interaction with highly metastatic prostate cancer cells (PC3M). We therefore postulate that HSkMC MVs and possibly exosomes may contain certain protein factor(s) that could be the cause of the cytotoxic effect observed on targeted tumour cells.Peer reviewedFinal Accepted Versio

Isolation of microvesicles and exosomes by microfiltration and estimation of normal reference range in blood plasma.

Current protocols for the isolation of microvesicles (MVs) and exosomes, which in the main focus on differential centrifugation, vary considerably (1). In an attempt to set a new standard, we describe a filtration protocol for isolating phosphatidylserine-positive MVs (larger than 100 nm in diameter) and exosomes. The key preparative step to successfully isolate both MVs and exosomes to a high degree of purity was a gentle sonication to break up exosome clumps. Filtration through a 100 nm pore size Millipore filter allowed for collection of exosomes in the filtrate. The larger MVs could then be recovered from the filter. Annexin V-PE MVs were sized and quantified using Polysciences Polybead Microspheres (200 nm) and BDTrucount tubes, respectively on a FACS CaliburTM flow cytometer. The normal reference range from normal human donors was found to be 0.51-2.82 x105 MVs/ml. Freeze/thawing of samples had little effect on MV counts and with age MV levels seemed only marginally reduced. Fasting status also affected MV levels, appearing up to 3-fold higher in fasting individuals. Smokers had lower MV counts and nicotine reduced MV release from THP-1 cells.Peer reviewedFinal Accepted Versio

Microvesicles and epithelial mesenchymal transition in the development of cancer

Microvesicles released by tumour cell lines are thought to play an important role in both extracellular matrix (ECM) invasion and evasion of the immune system. We have examined the role of MVs derived from a T cell line (Jurkat) on PNT2 cells (normal prostate cells) to see if they carry some bioactive molecules capable of inducing an epithelial to mesenchymal transition (EMT) in normal prostate cells. The link between EMT and malignancy has been well documented in almost all carcinomas of epithelial origin. However, to understand the mechanism by which MVs may induce this and to show the factors carried by MVs, further tests including invasion assays, angiogenesis and apoptotic assays as well as proteomic analyses will be needed. By microscopic analysis, PNT2 cells, which are of epithelial origin, treated with Jurkat MVs, showed some morphological changes. They become elongated, motile and morphologically mesenchymal-like as previously documented. The molecular changes were confirmed by immunohistochemical techniques using the fluorescencent microscope and flow cytometer. The experimental (MV-treated cells) compared to control (untreated cells) expressed high level of mesenchymal markers such as Vimentin and low levels of epithelial markers such as E-Cadherin. Proteins from PNT2 control cells and MV- treated cells were profiled by SDS- PAGE, MV-treated cells showing a band around 13 kDa. To identify this protein we intend to sequence it using mass spectrometry.Peer reviewedFinal Accepted Versio

Complement C2 receptor inhibitor trispanning: from man to schistosome

Horizontal gene transfer (HGT), in relation to genetic transfer between hosts and parasites, is a little described mechanism. Since the complement inhibitor CRIT was first discovered in the human Schistosoma parasite (the causative agent of Bilharzia) and in Trypanosoma cruzi (a parasite causing Chagas' disease), it has been found to be distributed amongst various species, ranging from the early teleost cod to rats and humans. In terms of evolutionary distance, as measured in a phylogenetic analysis of these CRIT genes at nucleotide level, the parasitic species are as removed from their human host as is the rat sequence, suggesting HGT. The hypotheses that CRIT in humans and schistosomes is orthologous and that the presence of CRIT in schistosomes occurs as a result of host-to-parasite HGT are presented in the light of empirical data and the growing body of data on mobile genetic elements in human and schistosome genomes. In summary, these data indicate phylogenetic proximity between Schistosoma and human CRIT, identity of function, high nucleotide/amino acid identity and secondary protein structure, as well as identical genomic organizatio

Complement-mediated extracellular vesicle release as a measure of endothelial dysfunction and prognostic marker for COVID-19 in peripheral blood - letter to the Editor

The recent articles in Clin. Hemorheol. Microcirc. by Jung et al. [1–3], elegantly highlighted the thrombotic complications that arise in severe coronavirus disease 2019 (COVID-19) and discussed the role vascular injury and associated hyper inflammation play in bringing about multi-organ failure in severe disease. Coagulation and venous thromboembolism (VTE) in COVID-19 commonly presents as deep vein thrombosis (DVT) or pulmonary embolism (PE), and occurs because of inflammation, blood vessel injury and associated endothelial dysfunction. Likely contributors to this thrombotic milieu, hitherto little discussed in this COVID-19 pandemic, include Extracellular Vesicles (EVs), nanosized, cell-derived intercellular communicative vesicles, carrying proteins, bioactive lipids and miRNAs. Endothelial cell- (EC-) derived EVs (EEVs) are often released because of endothelial injury [4] and also likely to contribute to this prothrombotic environment. Whilst EVs and VTE in cancer has been much described, there is a significant knowledge gap concerning EVs and VTE in infectious disease. This letter considers how, as part of ongoing inflammation, complement may be activated in SARS-CoV-2 infection and so mediate EV biogenesis. It also assesses the role procoagulant EVs play in the context of coagulopathy and VTE in COVID-19, and their potential as a prognostic peripheral blood marker

Decoy ACE2-expressing extracellular vesicles that competitively bind SARS-CoV-2 as a possible COVID-19 therapy

© 2020 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY-NC-ND - https://creativecommons.org/licenses/by-nc-nd/4.0/).The novel strain of coronavirus that appeared in 2019, SARS-CoV-2, is the causative agent of severe respiratory disease, COVID-19, and the ongoing pandemic. As for SARS-CoV that caused the SARS 2003 epidemic, the receptor on host cells that promotes uptake, through attachment of the spike (S) protein of the virus, is angiotensin-converting enzyme 2 (ACE2). In a recent article published by Batlle et al. (Clin. Sci. (Lond.) (2020) 134, 543-545) it was suggested that soluble recombinant ACE2 could be used as a novel biological therapeutic to intercept the virus, limiting the progression of infection and reducing lung injury. Another way, discussed here, to capture SARS-CoV-2, as an adjunct or alternative, would be to use ACE2+-small extracellular vesicles (sEVs). A competitive inhibition therapy could therefore be developed, using sEVs from engineered mesenchymal stromal/stem cells (MSCs), overexpressing ACE2.Peer reviewedFinal Published versio

Animal Models of Human Disease

© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).The use of animal models of human disease is critical for furthering our understanding of disease mechanisms, for the discovery of novel targets for treatment, and for translational research. This Special Topic entitled “Animal Models of Human Disease” aimed to collect state-of-the-art primary research studies and review articles from international experts and leading groups using animal models to study human diseases. Submissions were welcomed on a wide range of animal models and pathologies, including infectious disease, acute injury, regeneration, cancer, autoimmunity, degenerative and chronic disease. Seven participating MDPI journals supported the Special Topic, namely: Biomedicines, Cells, Current Issues in Molecular Biology, Diagnostics, Genes, the International Journal of Molecular Sciences, and the International Journal of Translational Medicine. In total, 46 papers were published in this Special Topic, with 37 full length original research papers, 2 research communications and 7 reviews. These contributions cover a wide range of clinically relevant, translatable, and comparative animal models, as well as furthering understanding of fundamental sciences, covering topics on physiological processes, on degenerative, inflammatory, infectious, autoimmune, neurological, metabolic, heamatological, hormonal and mitochondrial disorders, developmental processes and diseases, cardiology, cancer, trauma, stress, and ageing.Peer reviewe