Extracellular vesicles (EVs) are gaining ground as next-generation drug delivery modalities. Genetic fusion of the protein of interest to a scaffold protein with high EV-sorting ability represents a ...robust cargo loading strategy. To address the paucity of such scaffold proteins, we leverage a simple and reliable assay that can distinguish intravesicular cargo proteins from surface- as well as non-vesicular proteins and compare the EV-sorting potential of 244 candidate proteins. We identify 24 proteins with conserved EV-sorting abilities across five types of producer cells. TSPAN2 and TSPAN3 emerge as lead candidates and outperform the well-studied CD63 scaffold. Importantly, these engineered EVs show promise as delivery vehicles in cell cultures and mice as demonstrated by efficient transfer of luminal cargo proteins as well as surface display of different functional entities. The discovery of these scaffolds provides a platform for EV-based engineering.
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Extracellular vesicles (EVs) are phospholipid bilayer enclosed vesicles which play an important role in intercellular communication. To date, many studies have focused on therapeutic ...application of EVs. However, to progress EV applications faster towards the clinic, more information about the physical stability and scalable production of EVs is needed. The goal of this study was to evaluate EV recovery and function after varying several conditions in the isolation process or during storage.
Physical stability and recovery rates of EVs were evaluated by measuring EV size, particle and protein yields using nanoparticle tracking analysis, microBCA protein quantification assay and transmission electron microscopy. Western blot analyses of specific EV markers were performed to determine EV yields and purity. EV functionality was tested in an endothelial cell wound healing assay.
Higher EV recovery rates were found when using HEPES buffered saline (HBS) as buffer compared to phosphate buffered saline (PBS) during EV isolation. When concentrating EVs, 15 ml spinfilters with a 10 kDa membrane cutoff gave the highest EV recovery. Next, EV storage in polypropylene tubes was shown to be superior compared to glass tubes. The use of protective excipients during EV storage, i.e. bovine serum albumin (BSA) and Tween 20, improved EV preservation without influencing their functionality. Finally, it was shown that both 4 °C and −80 °C are suitable for short term storage of EVs. Together, our results indicate that optimizing buffer compositions, concentrating steps, protective excipients and storage properties may collectively increase EV recovery rates significantly while preserving their functional properties, which accelerates translation of EV-based therapeutics towards clinical application.
Langerhans cell histiocytosis (LCH) is a potentially life-threatening inflammatory myeloid neoplasia linked to paediatric neurodegeneration, whereby transformed LCH cells form agglomerated lesions in ...various organs. Although MAP-kinase pathway mutations have been identified in LCH cells, the functional consequences of these mutations and the mechanisms that cause the pathogenic behaviour of LCH cells are not well understood. In our study, we used an in vitro differentiation system and RNA-sequencing to compare monocyte-derived dendritic cells from LCH patients to those derived from healthy controls or patients with Crohn's disease, a non-histiocytic inflammatory disease. We observed that Interferon-γ treatment exacerbated intrinsic differences between LCH patient and control cells, including strikingly increased endoand exocytosis gene activity in LCH patients. We validated these transcriptional patterns in lesions and functionally confirmed that LCH cells exhibited increased endo- and exocytosis. Furthermore, RNA-sequencing of extracellular vesicles (EV) revealed the enrichment of pathological transcripts involved in cell adhesion, MAP-kinase pathway, vesicle trafficking and T-cell activation in LCH patients. Thus, we tested the effect of the LCH secretome on lymphocyte activity and found significant activation of NK cells. These findings implicate EVs in the pathology of LCH for the first time, in line with their established roles in the formation of various other tumour niches. Thus, we describe novel traits of LCH patient cells and suggest a pathogenic mechanism of potential therapeutic and diagnostic importance.
Summary
Reasons for low in vitro fertilisation rates in the horse include the difficulties in inducing capacitation and/or hyperactivation of stallion spermatozoa. The aim of this study was to ...analyse the effect of noncapacitating and capacitating modified Whitten’s (MW) and modified Tyrode’s medium (MT) and treatment with procaine (5 mmol), pentoxifylline (3.5 mmol) and trolox (120 mmol) on motility (CASA), capacitation, acrosomal status, viability and mitochondrial membrane potential of stallion spermatozoa (n = 4). While there was no influence of MW and MT on sperm motility, a significant increase in the percentage of viable‐capacitated spermatozoa was observed after incubation in capacitating MW (P < 0.05). Pentoxifylline showed no significant effect on the motility pattern but increased the proportion of live‐capacitated spermatozoa (P < 0.05). Trolox had no detectable effect on either capacitation or hyperactivation. Procaine was the only agent that induced hyperactivation in terms of a reduced proportion of progressively motile spermatozoa, straight line velocity, straightness, linearity and beat‐cross frequency and an increase in the amplitude of lateral head displacement (P < 0.05). The combination of capacitating Whitten’s medium and procaine showed the best results for the induction of capacitation and hyperactivation in stallion spermatozoa; this was possible even after short‐term incubation.
Neonatal and infant immune responses are characterized by a limited capability to generate protective Ab titers and memory B cells as seen in adults. Multiple studies support an immature or even ...impaired character of umbilical cord blood (UCB) B cells themselves. In this study, we provide a comprehensive molecular and functional comparison of B cell subsets from UCB and adult peripheral blood. Most UCB B cells have a mature, naive B cell phenotype as seen in adults. The UCB Ig repertoire is highly variable but interindividually conserved, as BCR clonotypes are frequently shared between neonates. Furthermore, UCB B cells show a distinct transcriptional program that confers accelerated responsiveness to stimulation and facilitated IgA class switching. Stimulation drives extensive differentiation into Ab-secreting cells, presumably limiting memory B cell formation. Humanized mice suggest that the distinctness of UCB versus adult B cells is already reflected by the developmental program of hematopoietic precursors, arguing for a layered B-1/B-2 lineage system as in mice, albeit our findings suggest only partial comparability to murine B-1 cells. Our study shows that UCB B cells are not immature or impaired but differ from their adult mature counterpart in a conserved BCR repertoire, efficient IgA class switching, and accelerated, likely transient response dynamics.
Mesenchymal stem or stromal cells (MSCs) are widely applied in regenerative and immune modulating therapies. Initially, it was assumed that administered MSCs integrate into damaged tissues to exert ...their clinical functions. However, in recent years accumulating evidence has been provided that MSCs rather act in a paracrine than a cellular manner. To this end, extracellular vesicles (EVs), which are secreted by MSCs in vivo and in vitro, seem to exert the MSCs’ therapeutic effects. So far, MSC‐EVs have been shown to improve functional recoveries following ischemic events, such as stroke and myocardial infarction, and to suppress symptoms in inflammatory diseases, such as in Graft‐versus‐Host disease (GvHD). Here, we summarize and discuss some current aspects regarding the therapeutic potential of MSC‐EVs.
ABSTRACTNeuron‐derived exosomes (NDEs) were enriched by anti‐L1CAM antibody immunoabsorption from plasmas of subjects ages 18–26 yr within 1 wk after a sports‐related mild traumatic brain injury ...(acute mTBI) (n = 18), 3 mo or longer after the last of 2–4 mTBIs (chronic mTBI) (n = 14) and with no recent history of TBI (controls) (n = 21). Plasma concentrations of NDEs, assessed by counts and levels of extracted exosome marker CD81, were significantly depressed by a mean of 45% in acute mTBI (P < 0.0001), but not chronic mTBI, compared with controls. Mean CD81‐normalized NDE levels of a range of functional brain proteins were significantly abnormal relative to those of controls in acute but not chronic mTBI, including ras‐related small GTPase 10, 73% decrease; annexin VII, 8.8‐fold increase; ubiquitin C‐terminal hydrolase L1, 2.5‐fold increase; AII spectrin fragments, 1.9‐fold increase; claudin‐5, 2.7‐fold increase; sodium‐potassium‐chloride cotransporter‐1, 2.8‐fold increase; aquaporin 4, 8.9‐fold increase (3.6‐fold increase in chronic mTBI); and synaptogyrin‐3, 3.1‐fold increase (1.3‐fold increase in chronic mTBI) (all acute mTBI proteins P < 0.0001). In chronic mTBI, there were elevated CD81‐normalized NDE levels of usually pathologic β‐amyloid peptide 1‐42 (1.6‐fold, P < 0.0001), P‐T181‐tau (2.2‐fold, P < 0.0001), P‐S396‐tau (1.6‐fold, P < 0.01), IL‐6 (16‐fold, P < 0.0001), and prion cellular protein (PRPc) (5.1‐fold, P < 0.0001) with lesser or greater (IL‐6, PRPc) increases in acute mTBI. Increases in NDE levels of most neurofunctional proteins in acute, but not chronic, mTBI, and elevations of most NDE neuropathological proteins in chronic and acute mTBI delineated phase‐specificity. Longitudinal studies of more mTBI subjects may identify biomarkers predictive of and etiologically involved in mTBI‐induced neurodegeneration.—Goetzl, E. J., Elahi, F. M., Mustapic, M., Kapogiannis, D., Pryhoda, M., Gilmore, A., Gorgens, K. A., Davidson, B., Granholm, A.‐C., Ledreux, A. Altered levels of plasma neuron‐derived exosomes and their cargo proteins characterize acute and chronic mild traumatic brain injury. FASEB J. 33, 5082–5088 (2019). www.fasebj.org
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