Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.

Upon cytokine treatment, members of the signal transducers and activators of transcription (STAT) family of proteins are phosphorylated on tyrosine and serine sites within the carboxyl-terminal region in cells. We show that in response to cytokine treatment, Stat3 is also acetylated on a single lysine residue, Lys685. Histone acetyltransferase p300-mediated Stat3 acetylation on Lys685 was reversible by type I histone deacetylase (HDAC). Use of a prostate cancer cell line (PC3) that lacks Stat3 and PC3 cells expressing wild-type Stat3 or a Stat3 mutant containing a Lys685-to-Arg substitution revealed that Lys685 acetylation was critical for Stat3 to form stable dimers required for cytokine-stimulated DNA binding and transcriptional regulation, to enhance transcription of cell growth-related genes, and to promote cell cycle progression in response to treatment with oncostatin M.

The transcriptional activator nuclear factor kappa B (NF-B) is required for the upregulation of a large number of genes in response to inflammation, viral and bacterial infection, and other stress stimuli. Genes that respond to NF-B encode a variety of cytokines, cell adhesion molecules, and acute-phase response proteins as well as apoptotic suppressor and effector proteins. It is believed that this reprogramming of gene expression is essential for cell survival during situations of physiological crisis (61). The activation of NF-B in response to stimulation by the proinflammatory cytokines tumor necrosis factor alpha (TNF-␣) and interleukin 1 beta (IL-1␤) has been extensively studied (17, 30); however, the mechanisms that modulate and eventually limit these responses are still poorly understood (61).We report here that the recently discovered protein kinase inhibitor protein RKIP (Raf kinase inhibitor protein) acts to inhibit NF-B activation. RKIP was first identified as an interacting partner of Raf-1 and shown to function as a negative regulator of the mitogen-activated protein (MAP) kinase (MAPK) cascade initiated by 76). The Raf-1-initiated pathway is comprised of three sequentially acting protein kinases: a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAPK. This basic relationship has now been found to be conserved in several protein kinase pathways. In the Raf-1 pathway the MAPK is ERK1/2 (extracellular signal-regulated kinase 1 and 2), the MAPKK is MEK1 (MAP/ERK kinase 1), and the MAPKKK is Raf-1 itself. Functional studies using both gain-of-function and loss-of-function approaches demonstrated that RKIP disrupts the interaction between 76). Depletion of endogenous RKIP upregulated Raf-1 kinase activity and MAPK signaling, whereas ectopic expression of RKIP suppressed Raf-1 kinase activity and MAPK signaling as well as v-Raf-mediated transformation. Biochemical studies showed that RKIP efficiently dissociated preformed Raf/MEK complexes and behaved kinetically as a competitive inhibitor of MEK phosphorylation. In vivo, the association of endogenous RKIP with Raf-1 correlated inversely with Raf-1 kinase activity during serum stimulation of quiescent cells.Active NF-B is a dimer that can be assembled from several members of the Rel family of transcription factors, and some form of NF-B is expressed in most cell types (61). In unstimulated cells, NF-B is retained in the cytoplasm in an inactive form bound to a family of inhibitory proteins known as IB (inhibitors of B). Activation of NF-B requires the phosphorylation and degradation of IB, which allows the NF-B dimer to translocate into the nucleus. Virtually all of the many stimuli that can activate NF-B cause the phosphorylation of IB on

While the role of nuclear transcription factor activator protein-1 (AP-1) in cell proliferation, and of nuclear factor-kappaB (NF-kappaB) in the suppression of apoptosis are known, their role in survival of prostate cancer cells is not well understood. We investigated the role of NF-kappaB and AP-1 in the survival of human androgen-independent (DU145) and -dependent (LNCaP) prostate cancer cell lines. Our results show that the faster rate of proliferation of DU145 cells when compared to LNCaP cells correlated with the constitutive expression of activated NF-kappaB and AP-1 in DU-145 cells. The lack of constitutive expression of NF-kappaB and AP-1 in LNCaP cells also correlated with their sensitivity to the antiproliferative effects of tumor necrosis factor (TNF). TNF induced NF-kappaB activation but not AP-1 activation in LNCaP cells. In DU145 cells both c-Fos and c-Jun were expressed and treatment with TNF activated c-Jun NH2-terminal kinase (JNK), needed for AP-1 activation. In LNCaP cells, however, only low levels of c-Jun was expressed and treatment with TNF minimally activated JNK. Treatment of cells with curcumin, a chemopreventive agent, suppressed both constitutive (DU145) and inducible (LNCaP) NF-kappaB activation, and potentiated TNF-induced apoptosis. Curcumin alone induced apoptosis in both cell types, which correlated with the downregulation of the expression of Bcl-2 and Bcl-xL and the activation of procaspase-3 and procaspase-8. Overall, our results suggest that NF-kappaB and AP-1 may play a role in the survival of prostate cancer cells, and curcumin abrogates their survival mechanisms.

Mesenchymal stem cells (MSCs) contribute to the recovery of tissue injury, providing a paracrine support. Cell-derived extracellular vesicles (EVs), carrying membrane and cytoplasmatic constituents of the cell of origin, have been described as a fundamental mechanism of intercellular communication. We previously demonstrated that EVs derived from human MSCs accelerated recovery following acute kidney injury (AKI) in vivo. The aim of the present study was to investigate the biodistribution and the renal localization of EVs in AKI. For this purpose, two methods for EV labeling suitable for in vivo tracking with optical imaging (OI), were employed using near infrared (NIR) dye (DiD): i) labeled EVs were generated by MSCs pre-incubated with NIR dye and collected from cell supernatants; ii) purified EVs were directly labeled with NIR dye. EVs obtained with these two procedures were injected intravenously (i.v.) into mice with glycerol-induced AKI and into healthy mice to compare the efficacy of the two labeling methods for in vivo detection of EVs at the site of damage. We found that the labeled EVs accumulated specifically in the kidneys of the mice with AKI compared with the healthy controls. After 5 h, the EVs were detectable in whole body images and in dissected kidneys by OI with both types of labeling procedures. The directly labeled EVs showed a higher and brighter fluorescence compared with the labeled EVs produced by cells. The signal generated by the directly labeled EVs was maintained in time, but provided a higher background than that of the labeled EVs produced by cells. The comparison of the two methods indicated that the latter displayed a greater specificity for the injured kidney.

The very process of deregulated oncogene expression during cancer development also sensitizes cancer cells to apoptotic signals (1-3). Deregulated oncoproteins such as E1a and c-Myc promote apoptosis by activating multiple downstream proapoptotic effector pathways (4, 5). Additional mechanisms of sensitizing cancer cells to apoptosis by an activated oncoprotein have been described (6, 7). For example, E2F sensitized cells to apoptosis through down-regulation of anti-apoptotic signals (7). Here we show that cancer cells can also be sensitized to apoptosis by up-regulating the expression levels of RKIP (Raf kinase inhibitor protein). RKIP was originally identified as an interacting partner of Raf-1 and a negative regulator of the mitogen-activated protein kinase cascade initiated by Raf-1 (8). RKIP also inhibits nuclear factor B (NF-B)1 signaling by negatively modulating the activating phosphorylation of IKK␣ and IKK␤ via upstream kinases (9). Although the molecular mechanism by which RKIP inhibits the Raf and NF-B signaling pathways has been partially delineated, little is known about the biological relevance of the inhibition of these pathways by RKIP. In addition to these functions, we presently demonstrate the rapid up-regulation of RKIP during induction of chemotherapy-triggered apoptosis in human prostate and breast cancer cells. However, in DNA-damaging agent-resistant cancer cells, treatment with the drugs does not up-regulate RKIP expression. Ectopic expression of RKIP sensitizes DNA damage agentresistant cells to undergo apoptosis. Down-regulation of RKIP expression confers resistance to 9-nitrocamptothecin (9NC) by releasing its inhibitory constraint on two major survival pathways in cancer cells. Our studies suggest that RKIP represents a novel apoptotic marker in human cancer cells. MATERIALS AND METHODSCell Lines, Plasmid Constructs, and Chemicals-The human breast cell lines 578T and 578Bst were purchased from American Type Culture Collection (Manassas, VA). A human breast cancer MCF7 cell subline resistant to 9NC treatment was a gift from Dr. Ray Frackelton (Brown University). The human prostate cell lines LNCaP, DU145, and PC3 were purchased from American Type Culture Collection. Early (Ͻ30)-or late (Ͼ100)-passage DU145 cells were not used for this study. The 9NC-resistant DU145 cell subline, RC1, was established by continuous exposure of DU145 cells to 9NC (10). All cell lines were grown in conditions suggested by American Type Culture Collection. MCF7 and

Mesenchymal stromal cells (MSC) have been shown to reverse radiation damage to marrow stem cells. We have evaluated the capacity of MSC-derived extracellular vesicles (MSC-EVs) to mitigate radiation injury to marrow stem cells at 4 hours to 7 days after irradiation. Significant restoration of marrow stem cell engraftment at 4, 24 and 168 hours post-irradiation by exposure to MSC-EVs was observed at 3 weeks to 9 months after transplant and further confirmed by secondary engraftment. Intravenous injection of MSC-EVs to 500cGy exposed mice led to partial recovery of peripheral blood counts and restoration of the engraftment of marrow. The murine hematopoietic cell line, FDC-P1 exposed to 500 cGy, showed reversal of growth inhibition, DNA damage and apoptosis on exposure to murine or human MSC-EVs. Both murine and human MSC-EVs reverse radiation damage to murine marrow cells and stimulate normal murine marrow stem cell/progenitors to proliferate. A preparation with both exosomes and microvesicles was found to be superior to either microvesicles or exosomes alone. Biologic activity was seen in freshly isolated vesicles and in vesicles stored for up to 6 months in 10% DMSO at −80°C. These studies indicate that MSC-EVs can reverse radiation damage to bone marrow stem cells.

Rituximab (Rituxan, IDEC-C2B8) has been shown to sensitize nonHodgkin's lymphoma (NHL) cell lines to chemotherapeutic drug-induced apoptosis. Rituximab treatment of Bcl-2-deficient Ramos cells and Bcl-2-expressing Daudi cells selectively decreases Bcl-xL expression and sensitizes the cells to paclitaxel-induced apoptosis. This study delineates the signaling pathway involved in rituximab-mediated Bcl-xL down-regulation in Ramos and Daudi NHL B cells. We hypothesized that rituximab may interfere with the extracellular signal-regulated kinase (ERK) 1/2 pathway, leading to decreased Bcl-xL expression. Rituximab (20 g/mL) inhibited the kinase activity of mitogen-activated protein kinase kinase (MEK) 1/2 and reduced the phosphorylation of the components of the ERK1/2 pathway (Raf-1, MEK1/2, and ERK1/2) and decreased activator protein-1 DNA binding activity and Bcl-xL gene expression. These events occurred with similar kinetics and were observed 3 to 6 hours after rituximab treatment. Rituximab-mediated effects were corroborated by using specific inhibitors of the ERK1/2 pathway, which also reduced Bcl-xL levels and sensitized the NHL B cells to paclitaxel-induced apoptosis. Previous findings implicated a negative regulatory role of the Raf-1 kinase inhibitor protein (RKIP) on the ERK1/2 pathway. Rituximab treatment of NHL B cells significantly up-regulated RKIP expression, thus interrupting the ERK1/2 signaling pathway through the physical association between Raf-1 and RKIP, which was concomitant with Bcl-xL downregulation. These novel findings reveal a signaling pathway triggered by rituximab, whereby rituximab-mediated up-regulation of RKIP adversely regulates the activity of the ERK1/2 pathway, Bcl-xL expression, and subsequent chemosensitization of drug-refractory NHL B cells. The significance of these findings is discussed.