No abstract

Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull-down, and OptiPrep(TM) density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep(TM) density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active.

The endoglycosidase heparanase is an important in the degradation of the extracellular matrix by invading cells, notably metastatic tumor cells and migrating leukocytes. Here we report the cDNA sequence of the human platelet enzyme, which encodes a unique protein of 543 amino acids, and the identification of highly homologous sequences in activated mouse T cells and in a highly metastatic rat adenocarcinoma. Furthermore, the expression of heparanase mRNA in rat tumor cells correlates with their metastatic potential. Exhaustive studies have shown only one heparanase sequence, consistent with the idea that this enzyme is the dominant endoglucuronidase in mammalian tissues.

Summary Histidine-rich glycoprotein (HRG) is an abundant plasma glycoprotein that has a multidomain structure, interacts with many ligands, and has been shown to regulate a number of important biological processes. HRG ligands include Zn 2+ and haem, tropomyosin, heparin and heparan sulphate, plasminogen, plasmin, fibrinogen, thrombospondin, IgG, Fc γ R and complement. In many cases, the histidine-rich region of the molecule enhances ligand binding following interaction with Zn 2+ or exposure to low pH, conditions associated with sites of tissue injury or tumour growth. The multidomain nature of HRG indicates that it can act as an extracellular adaptor protein, bringing together disparate ligands, particularly on cell surfaces. HRG binds to most cells primarily via heparan sulphate proteoglycans, binding which is also potentiated by elevated free Zn 2+ levels and low pH. Recent reports have shown that HRG can modulate angiogenesis and additional studies have shown that it may regulate other physiological processes such as cell adhesion and migration, fibrinolysis and coagulation, complement activation, immune complex clearance and phagocytosis of apoptotic cells. This review outlines the molecular, structural, biological and clinical properties of HRG as well as describing the role of HRG in various physiological processes.

Phagocytosis serves as one of the key processes involved in development, maintenance of tissue homeostasis, as well as in eliminating pathogens from an organism. Under normal physiological conditions, dying cells (e.g., apoptotic and necrotic cells) and pathogens (e.g., bacteria and fungi) are rapidly detected and removed by professional phagocytes such as macrophages and dendritic cells (DCs). In most cases, specific receptors and opsonins are used by phagocytes to recognize and bind their target cells, which can trigger the intracellular signalling events required for phagocytosis. Depending on the type of target cell, phagocytes may also release both immunomodulatory molecules and growth factors to orchestrate a subsequent immune response and wound healing process. In recent years, evidence is growing that opsonins and receptors involved in the removal of pathogens can also aid the disposal of dying cells at all stages of cell death, in particular plasma membrane-damaged cells such as late apoptotic and necrotic cells. This review provides an overview of the molecular mechanisms and the immunological outcomes of late apoptotic/necrotic cell removal and highlights the striking similarities between late apoptotic/necrotic cell and pathogen clearance. The immune system is constantly under pressure to accurately distinguish foreign materials or pathogens (non-self) from normal healthy tissues (self), and make an appropriate immune response to non-self molecules through a range of effector mechanisms. It is equally important for the immune system to distinguish healthy viable cells (self) from dying cells (altered-self) during the course of tissue remodelling or tissue injury to prevent the release of intracellular molecules that may damage neighbouring cells or stimulate an immunogenic response against self (i.e., an autoimmune response). Professional phagocytes of the innate immune system use a broad range of germline-encoded receptors and opsonins to discriminate viable cells from pathogens and dying cells, and aid the removal of non-self and altered-self through phagocytosis.1,2 Not surprisingly, impairment of phagocytosis due to a deficiency in key phagocytic components such as C1q, one of the first components of the classical complement cascade, has been implicated in an increased susceptibility to bacterial infection 3 as well as in the development of autoimmune diseases such as systemic lupus erythematosus (SLE).4 Therefore, understanding the molecular mechanisms of phagocytosis will provide new insights into numerous physiological and pathological processes.Under normal physiological conditions, cells of a multicellular organism predominantly die through the well-defined process known as apoptosis or programmed cell death (see Elmore 5 for an extensive review). During different stages of apoptotic cell death, a precise set of morphological and These authors contributed equally to this work.