Nanovesicles released by OKT3 hybridoma express fully active antibodies.

Recent findings have shown that nanovesicles preparations from either primary immune cells culture supernatants or plasma contain immunoglobulins, suggesting that a natural way of antibody production may be through exosome release. To verify this hypothesis, we used the OKT3 hybridoma clone, which produces a murine IgG2a monoclonal antibody used to reduce rejection in patients undergoing organ transplantation. We showed exosome-associated immunoglobulins in hybridoma supernatants, by Western blot, nanoscale flow cytometry and immunocapture-based ELISA. The OKT3-exo was also being able to trigger cytokines production in both CD4 and CD8 T cells. These results show that nanovesicles contain immunoglobulin and could be used for immunotherapy. These data could lead to a new approach to improve the effectiveness of therapeutic antibodies by exploiting their natural property to be expressed on nanovesicle membrane, that probably render them more stable and as a consequence more capable to interact with their specific ligand in the best way.

Exosome levels in human body fluids: A tumor marker by themselves?

Despite considerable research efforts, the finding of reliable tumor biomarkers remains challenging and unresolved. In recent years a novel diagnostic biomedical tool with high potential has been identified in extracellular nanovesicles or exosomes. They are released by the majority of the cells and contain detailed molecular information on the cell of origin including tumor hallmarks. Exosomes can be isolated from easy accessible body fluids, and most importantly, they can provide several biomarkers, with different levels of specificity. Recent clinical evidence shows that the levels of exosomes released into body fluids may themselves represent a predictive/diagnostic of tumors, discriminating cancer patients from healthy subjects. The aim of this review is to highlight these latest challenging findings to provide novel and groundbreaking ideas for successful tumor early diagnosis and follow-up.

Reprint of "EXOSOME LEVELS IN HUMAN BODY FLUIDS: A TUMOR MARKER BY THEMSELVES?"

Despite considerable research efforts, the finding of reliable tumor biomarkers remains challenging and unresolved. In recent years a novel diagnostic biomedical tool with high potential has been identified in extracellular nanovesicles or exosomes. They are released by the majority of the cells and contain detailed molecular information on the cell of origin including tumor hallmarks. Exosomes can be isolated from easy accessible body fluids, and most importantly, they can at once provide with several biomarkers, with different levels of specificity. Recent clinical evidence shows that the levels of exosomes released into body fluids may by themselves represent a predictive/diagnostic of tumors, discriminating cancer patients from healthy subjects. The aim of this review is to highlight these latest challenging findings to provide novel and groundbreaking ideas for successful tumor early diagnosis and follow-up.

Physical, chemical and kinetic factors affecting prion infectivity.

The mouse-adapted scrapie prion strain RML is one of the most widely used in prion research. The introduction of a cell culture-based assay of RML prions, the scrapie cell assay (SCA) allows more rapid and precise prion titration. A semi-automated version of this assay (ASCA) was applied to explore a range of conditions that might influence the infectivity and properties of RML prions. These include resistance to freeze-thaw procedures; stability to endogenous proteases in brain homogenate despite prolonged exposure to varying temperatures; distribution of infective material between pellet and supernatant after centrifugation, the effect of reducing agents and the influence of detergent additives on the efficiency of infection. Apparent infectivity is increased significantly by interaction with cationic detergents. Importantly, we have also elucidated the relationship between the duration of exposure of cells to RML prions and the transmission of infection. We established that the infection process following contact of cells with RML prions is rapid and followed an exponential time course, implying a single rate-limiting process.

A systematic investigation of production of synthetic prions from recombinant prion protein.

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre 'synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20,000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved.

The regulation of exosome function in the CNS: implications for neurodegeneration.

Exosomes are nanovesicles, generally 50 to 90 nm in diameter, that correspond to the intraluminal vesicles of the endosomal multivesicular bodies and are secreted upon fusion of multivesicular bodies with the plasma membrane. Their molecular content is highly selected and includes not only specific proteins and lipids, but also RNA species, such as messenger RNAs (mRNAs) and microRNAs (miRNAs), which are delivered and active in target cells. As they are released in body fluids, exosomes can shuttle molecules for long distances. In the CNS they have been shown to regulate neuronal development and regeneration, and to modulate synaptic functions. In neurodegenerative diseases, they have an important role in propagating neurotoxic misfolded protein from one cell to another and, as recent data show, possibly other molecules contributing to neurotoxicity. Some exosomal lipids such as gangliosides GM1 and GM3 enhance the aggregation of alpha-synuclein, and RNA exosomal cargo is also altered during pathologies such as Alzheimer's disease, prion diseases and amyotrophic lateral sclerosis. The aim of this review is to focus on the regulation of CNS exosomal function and highlight pathways that might have a role in the neurodegenerative process. The identification of the novel exosomal molecules involved in neurodegenerative diseases could provide important insights into the pathogenesis and contribute to the finding of novel diagnostic biomarkers and therapeutic approaches.

Detection of cellular prion protein in exosomes derived from ovine plasma.

Prion protein (PrP) is present at extremely low levels in the blood of animals and its detection is complicated by the poor sensitivity of current standard methodologies. Interesting results have been obtained with recent advanced technologies that are able to detect minute amounts of the pathological PrP (PrPSc), but their efficiency is reduced by various factors present in blood. In this study, we were able to extract cellular PrP (PrPC) from plasma-derived exosomes by a simple, fast method without the use of differential ultracentrifugation and to visualize it by Western blotting, reducing the presence of most plasma proteins. This result confirms that blood is capable of releasing PrP in association with exosomes and could be useful to better study its role in the pathogenesis of transmissible spongiform encephalopathies.

Detection of exosomal prions in blood by immunochemistry techniques.

In most forms of prion diseases, blood is infectious, but detection by immunochemistry techniques of the only available marker of infection (the misfolded prion protein, PrPTSE) in blood remains elusive. We developed a novel method for the detection of PrPTSE in blood of prion-infected rodents based on the finding that PrPTSE is associated with plasma exosomes. However, further purification of the exosomes on a sucrose gradient was necessary to remove plasma immunoglobulins, which interfere with PrPTSE, masking its detection by immunochemistry. Finally, we report that about 20% of plasma infectivity is associated with exosomes.

Exosomes: the future of biomarkers in medicine.

Exosomes are nanovesicles secreted into the extracellular environment upon internal vesicle fusion with the plasma membrane. The molecular content of exosomes is a fingerprint of the releasing cell type and of its status. For this reason, and because they are released in easily accessible body fluids such as blood and urine, they represent a precious biomedical tool. A growing body of evidence suggests that exosomes may be used as biomarkers for the diagnosis and prognosis of malignant tumors. This article focuses on the exploitation of exosomes as diagnostic tools for human tumors and discusses possible applications of the same strategies to other pathologies, such as neurodegenerative diseases.

Identification of misfolded proteins in body fluids for the diagnosis of prion diseases.

Transmissible spongiform encephalopathy (TSE) or prion diseases are fatal rare neurodegenerative disorders affecting man and animals and caused by a transmissible infectious agent. TSE diseases are characterized by spongiform brain lesions with neuronal loss and the abnormal deposition in the CNS, and to less extent in other tissues, of an insoluble and protease resistant form of the cellular prion protein (PrP(C)), named PrP(TSE). In man, TSE diseases affect usually people over 60 years of age with no evident disease-associated risk factors. In some cases, however, TSE diseases are unequivocally linked to infectious episodes related to the use of prion-contaminated medicines, medical devices, or meat products as in the variant Creutzfeldt-Jakob disease (CJD). Clinical signs occur months or years after infection, and during this silent period PrP(TSE), the only reliable marker of infection, is not easily measurable in blood or other accessible tissues or body fluids causing public health concerns. To overcome the limit of PrP(TSE) detection, several highly sensitive assays have been developed, but attempts to apply these techniques to blood of infected hosts have been unsuccessful or not yet validated. An update on the latest advances for the detection of misfolded prion protein in body fluids is provided.

Analysis of combinatorial variability reveals selective accumulation of the fibronectin type III domains B and D of tenascin-C in injured brain.

Tenascin-C (Tnc) is a multimodular extracellular matrix glycoprotein that is markedly upregulated in CNS injuries where it is primarily secreted by reactive astrocytes. Different Tnc isoforms can be generated by the insertion of variable combinations of one to seven (in rats) alternatively spliced distinct fibronectin type III (FnIII) domains to the smallest variant. Each spliced FnIII repeat mediates specific actions on neurite outgrowth, neuron migration or adhesion. Hence, different Tnc isoforms might differentially influence CNS repair. We explored the expression pattern of Tnc variants after cortical lesions and after treatment of astrocytes with various cytokines. Using RT-PCR, we observed a strong upregulation of Tnc transcripts containing the spliced FnIII domains B or D in injured tissue at 2-4 days post-lesion (dpl). Looking at specific combinations, we showed a dramatic increase of Tnc isoforms harboring the neurite outgrowth-promoting BD repeat with both the B and D domains being adjacent to each other. Isoforms containing only the axon growth-stimulating spliced domain D were also dramatically enhanced after injury. Injury-induced increase of Tnc proteins comprising the domain D was confirmed by Western Blotting and immunostaining of cortical lesions. In contrast, the FnIII modules C and AD1 were weakly modulated after injury. The growth cone repulsive A1A2A4 domains were poorly expressed in normal and injured tissue but the smallest isoform, which is also repellant, was highly expressed after injury. Expression of the shortest Tnc isoform and of variants containing B, D or BD, was strongly upregulated in cultured astrocytes after TGFbeta1 treatment, suggesting that TGFbeta1 could mediate, at least in part, the injury-induced upregulation of these isoforms. We identified complex injury-induced differential regulations of Tnc isoforms that may well influence axonal regeneration and repair processes in the damaged CNS.

Heparan sulphate proteoglycans in glia and in the normal and injured CNS: expression of sulphotransferases and changes in sulphation.

Heparan sulphate proteoglycans (HSPGs) have multiple functions relevant to the control of the CNS injury response, particularly in modulating the effects of growth factors and localizing molecules that affect axon growth. We examined the pattern of expression and glycanation of HSPGs in the normal and damaged CNS, and in astrocytes and oligodendrocyte precursors because of their participation in the injury reaction. The composition of HS glycosaminoglycan (GAG) chains was analysed by biochemical analysis and by the binding of antibodies that recognize sulphated epitopes. We also measured levels of HS sulphotransferases and syndecans. Compared with oligodendrocytes, oligodendrocyte precursors have more 2-O-sulphation in their HS GAG. This is accompanied by higher expression of the enzyme responsible for 2-O-sulphation, HS 2-O-sulphotransferase (HS2ST) and a fall in syndecan-1. Astrocytes treated with tumour growth factor (TGF)alpha or TGFbeta to mimic the injury response showed upregulation of syndecan-1 and HS2ST correlating with an increase in 2-O-sulphate residues in their HS GAGs. This also correlated with increased staining with AO4B08 anti-GAG antibody that recognizes high sulphation, and reduced staining with RB4EA12 recognizing low sulphation. After injury to the adult rat brain there was an overall increase in the quantity of HSPG around the injury site, mRNA for HS2ST was increased, and the changes in staining with sulphation-specific antibodies were consistent with an increase in 2-O-sulphated HS. Syndecan-1 was upregulated in astrocytes. The major injury-related change, seen in injured brain and cultured glia, was an increase in 2-O-sulphated HS and increased syndecan-1, suggesting novel approaches to modulating scar formation.

Removal of the glycosylphosphatidylinositol anchor from PrP(Sc) by cathepsin D does not reduce prion infectivity.

According to the protein-only hypothesis of prion propagation, prions are composed principally of PrP(Sc), an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrP(C)), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrP(Sc) to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrP(C). Removal of the GPI anchor from PrP(Sc) requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrP(Sc) in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has no effect (i) on an in vitro amplification model of PrP(Sc), (ii) on the prion titre as determined by a highly sensitive N2a-cell based bioassay, or (iii) in a mouse bioassay. These results show that the GPI anchor has little or no role in either the propagation of PrP(Sc) or on prion infectivity.

Two separate metalloproteinase activities are responsible for the shedding and processing of the NG2 proteoglycan in vitro.

A high proportion of NG2 in the adult rat spinal cord is saline-soluble and migrates slightly faster than intact NG2 on SDS-PAGE, suggesting that it represents the shed ectodomain of NG2. In the injured cerebral cortex, much of the overall increase in NG2 is due to the saline-soluble (shed), rather than the detergent-soluble (intact), form. Hydroxamic acid metalloproteinase inhibitors, but not TIMPs, were able to prevent NG2 shedding in oligodendrocyte precursor cells (OPCs) in vitro. The generation of another truncated form of NG2 was, however, sensitive to TIMP-2 and TIMP-3. Two observations suggest that NG2 is involved in PDGF signaling in OPCs: the rate of NG2 shedding increased with cell density and NG2 expression was increased in the absence of PDGF. Ectodomain shedding converts NG2 into a diffusible entity able to interact with the growth cone, and we suggest that this release is likely to enhance its axon growth-inhibitory activity.

Chondroitin 6-sulphate synthesis is up-regulated in injured CNS, induced by injury-related cytokines and enhanced in axon-growth inhibitory glia.

Chondroitin sulphate proteoglycans (CSPGs) are up-regulated in the CNS after injury and inhibit axon regeneration mainly through their glycosaminoglycan (CS-GAG) chains. We have analysed the mRNA levels of the CS-GAG synthesizing enzymes and measured the CS-GAG disaccharide composition by chromatography and immunocytochemistry. Chondroitin 6-sulfotransferase 1 (C6ST1) is up-regulated in most glial types around cortical injuries, and its sulphated product CS-C is also selectively up-regulated. Treatment with TGFalpha and TGFbeta, which are released after brain injury, promotes the expression of C6ST1 and the synthesis of 6-sulphated CS-GAGs in primary astrocytes. Oligodendrocytes, oligodendrocyte precursors and meningeal cells are all inhibitory to axon regeneration, and all express high levels of CS-GAG, including high levels of 6-sulphated GAG. In axon growth-inhibitory Neu7 astrocytes C6ST1 and 6-sulphated GAGs are expressed at high levels, whereas in permissive A7 astrocytes they are not detectable. These results suggest that the up-regulation of CSPG after CNS injury is associated with a specific sulphation pattern on CS-GAGs, mediating the inhibitory properties of proteoglycans on axonal regeneration.

Proteoglycans and brain repair.

Proteoglycans are complex molecules composed of long, unbranched sugar chains attached to a protein core. In the mammalian central nervous system, they are a major component of the extracellular matrix and of the cellular surface. After a central nervous system injury, their expression in the lesion area changes strongly and contributes to the inhibition of axon regrowth and brain repair.

Regulation of RPTPbeta/phosphacan expression and glycosaminoglycan epitopes in injured brain and cytokine-treated glia.

Several chondroitin sulfate proteoglycans (CSPGs) are upregulated after CNS injury and are thought to limit axonal regeneration in the adult mammalian CNS. Therefore, we examined the expression of the CSPG, receptor protein tyrosine phosphatase beta (RPTPbeta)/phosphacan, after a knife lesion to the cerebral cortex and after treatment of glial cultures with regulatory factors. The three splice variants of this CSPG gene, the secreted isoform, phosphacan, and the two transmembrane isoforms, the long and short RPTPbeta, were examined. Western blot and immunostaining analysis of injured and uninjured tissue revealed a transient decrease of phosphacan protein levels, but not of short RPTPbeta, in the injured tissue from 1 to 7 days postlesion (dpl). By real time RT-PCR, we show that phosphacan and long RPTPbeta mRNA levels are transiently down-regulated at 2 dpl, unlike those of short RPTPbeta which increased after 4 dpl. In contrast to the core glycoprotein, the phosphacan chondroitin sulfate (CS) glycosaminoglycan epitope DSD-1 was up-regulated after 7 dpl. Phosphacan was expressed by cultivated astrocytes and oligodendrocyte precursors but was more glycanated in oligodendrocyte precursors, which produce more of DSD-1 epitope than astrocytes. Epidermal growth factor/transforming growth factor alpha strongly increased the astrocytic expression of long RPTPbeta and phosphacan and slightly the short RPTPbeta protein levels, while interferon gamma and tumor necrosis factor alpha reduced astrocytic levels of phosphacan, but not of the receptor forms. Examining the effects of phosphacan on axon growth from rat E17 cortical neurons, we found that phosphacan stimulates outgrowth in a largely CS dependent manner, while it blocks the outgrowth-promoting effects of laminin through an interaction that is not affected by removal of the CS chains. These results demonstrate complex injury-induced modifications in phosphacan expression and glycanation that may well influence axonal regeneration and repair processes in the damaged CNS.