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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. (C) 2016 Elsevier B.V. All rights reserved.

collaboration au projet H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD); International audience; Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions. Containing cell-type specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their physical functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clinical, academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and.Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.

Exosomes are extracellular nanovesicles primarily involved in the pathogenesis of many diseases including cancer. This study was set out from recent evidence that extracellular acidity may increase the exosome release by cancer cells. However, this preliminary evidence did not provide solid information on whether the pH-dependent exosome over-release represents a common feature of all cancers. To the purpose of demonstrating that cancer acidity is a major determinant in inducing an increased exosome release by human cancer cells, we evaluated human tumor cell lines deriving from either colon, breast, prostate cancers, melanoma, or osteosarcoma. All cell lines were cultured in either the current 7.4 pH or the typical pH of cancer that is 6.5. The levels of released extracellular vesicles were measured by protein counts, nanoparticle tracking analysis (NTA), and nanoscale flow cytometry. The results showed that pH 6.5 induced a remarkable increase in exosome release, and buffering the medium significantly reduced the exosome release in all cancers. With these results, we provide, for the first time, evidence that tumor acidity and exosome levels represent common cancer phenotypes.

Proton pumps like the vacuolar-type H+ ATPase (V-ATPase) are involved in the control of cellular pH in normal and tumor cells. Treatment with proton pump inhibitors (PPI) induces sensitization of cancer cells to chemotherapeutics via modifications of cellular pH gradients. It is also known that low pH is the most suitable condition for a full PPI activation. Here, we tested whether PPI treatment in unbuffered culture conditions could affect survival and proliferation of human B-cell tumors. First, we showed that PPI treatment increased the sensitivity to vinblastine of a pre-B acute lymphoblastic leukemia (ALL) cell line. PPI, per se, induced a dose-dependent inhibition of proliferation of tumor B cells, which was associated with a dose- and time-dependent apoptotic-like cytotoxicity in B-cell lines and leukemic cells from patients with pre-B ALL. The effect of PPI was mediated by a very early production of reactive oxygen species (ROS), that preceded alkalinization of lysosomal pH, lysosomal membrane permeabilization, and cytosol acidification, suggesting an early destabilization of the acidic vesicular compartment. Lysosomal alterations were followed by mitochondrial membrane depolarization, release of cytochrome c, chromatin condensation, and caspase activation. However, inhibition of caspase activity did not affect PPI-induced cell death, whereas specific inhibition of ROS by an antioxidant (N-acetylcysteine) significantly delayed cell death and protected both lysosomal and mitochondrial membranes. The proapoptotic activity of PPI was consistent with a clear inhibition of tumor growth following PPI treatment of B-cell lymphoma in severe combined immunodeficient mice. This study further supports the importance of acidity and pH gradients in tumor cell homeostasis and suggests new therapeutic approaches for human B-cell tumors based on PPI. [Cancer Res 2007;67(11):5408–17]

Background: Resistance to antitumor agents is a major cause of treatment failure in patients with cancer. Some mechanisms of tumor resistance to cytotoxic drugs may involve increased acidification of extracellular compartments. We investigated whether proton pump inhibitors (PPIs), currently used in the anti-acid treatment of peptic disease, could inhibit the acidification of the tumor microenvironment and increase the sensitivity of tumor cells to cytotoxic agents. Methods: We pretreated cell lines derived from human melanomas, adenocarcinomas, and lymphomas with the PPIs omeprazole, esomeprazole, or pantoprazole and tested their response to cytotoxic drugs in cell death assays. We also evaluated extracellular and intracellular pH and vacuolar-H + -ATPase (V-H + -ATPase) expression, distribution, and activity in PPI-pretreated cells by using western blot analyses, immunocytochemistry, laser scanning confocal analysis, and bioluminescence assays. Finally, we evaluated human melanoma growth and cisplatin sensitivity with or without omeprazole pretreatment in xenografted SCID/SCID mice. Results: PPI pretreatment sensitized tumor cell lines to the effects of cisplatin, 5-fluorouracil, and vinblastine, with an IC 50 value reduction up to 2 logs. PPI pretreatment was associated with the inhibition of V-H + -ATPase activity and increases in both extracellular pH and the pH of lysosomal organelles. PPI pretreatment induced a marked increase in the cytoplasmic retention of the cytotoxic drugs, with clear targeting to the nucleus in the case of doxorubicin. In in vivo experiments, oral pretreatment with omeprazole was able to induce sensitivity of human solid tumors to cisplatin. Conclusion: Our results open new possibilities for the treatment of drug-resistant tumors through combination strategies based on the use of well-tolerated pH modulators such as PPIs.

A major challenge of AIDS research is the development of therapeutic vaccine strategies capable of inducing the humoral and cellular arms of the immune responses against HIV-1. In this work, we evaluated the capability of DCs pulsed with aldrithiol-2–inactivated HIV-1 in inducing a protective antiviral human immune response in SCID mice reconstituted with human PBL (hu-PBL-SCID mice). Immunization of hu-PBL-SCID mice with DCs generated after exposure of monocytes to GM-CSF/IFN-α (IFN-DCs) and pulsed with inactivated HIV-1 resulted in a marked induction of human anti–HIV-1 antibodies, which was associated with the detection of anti-HIV neutralizing activity in the serum. This vaccination schedule also promoted the generation of a human CD8+ T cell response against HIV-1, as measured by IFN-γ Elispot analysis. Notably, when the hu-PBL-SCID mice immunized with antigen-pulsed IFN-DCs were infected with HIV-1, inhibition of virus infection was observed as compared with control animals. These results suggest that IFN-DCs pulsed with inactivated HIV-1 can represent a valuable approach of immune intervention in HIV-1–infected patients.

Since denditric cells (DC) represent the main players linking innate and adaptive immunity, their prompt generation from blood cells would be instrumental for an efficient immune response to infections. Consistent with this, CD2+ monocytes were found to express the DC maturation marker CD83, along with acquisition of high antigen-presenting activity, after a surprisingly short time in culture. This rapid process is associated with expression of IFN-alpha/beta genes and secretion of low levels of pro-inflammatory cytokines. Exposure of monocytes to IFN-alpha, but not to IL-4, induced persistence of CD2+/CD83+ cells, which were fully competent in stimulating primary responses by naive T cells. These results unravel the natural pathway by which infection-induced signals rapidly transform pre-armed monocytes into active DC.

Background We recently demonstrated that epicardial progenitor cells participate in the regenerative response to myocardial infarction (MI) and factors released in the pericardial fluid (PF) may play a key role in this process. Exosomes are secreted nanovesicles of endocytic origin, identified in most body fluids, which may contain molecules able to modulate a variety of cell functions. Here, we investigated whether exosomes are present in the PF and their potential role in cardiac repair. Methods and results Early gene expression studies in 3day-infarcted mouse hearts showed that PF induces epithelial-to-mesenchymal transition (EMT) in epicardial cells. Exosomes were identified in PFs from non-infarcted patients (PFC) and patients with acute MI (PFMI). A shotgun proteomics analysis identified clusterin in exosomes isolated from PFMI but not from PFC. Notably, clusterin has a protective effect on cardiomyocytes after acute MI in vivo and is an important mediator of TGFβ-induced. Clusterin addition to the pericardial sac determined an increase in epicardial cells expressing the EMT marker α-SMA and, interestingly, an increase in the number of epicardial cells ckit + /α-SMA + , 7days following MI. Importantly, clusterin treatment enhanced arteriolar length density and lowered apoptotic rates in the peri-infarct area. Hemodynamic studies demonstrated an improvement in cardiac function in clusterin-treated compared to untreated infarcted hearts. Conclusions Exosomes are present and detectable in the PFs. Clusterin was identified in PFMI-exosomes and might account for an improvement in myocardial performance following MI through a framework including EMT-mediated epicardial activation, arteriogenesis and reduced cardiomyocyte apoptosis.

The migration capability of dendritic cells (DCs) is regulated by their response to factors, namely chemokines, that characterize maturation stage and shape their functional activities. This study examines the morphology, expression of chemokines/chemokine receptors, and migration properties of DCs generated after treatment of monocytes with type I interferon (IFN) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (IFN-DCs). IFN-DCs showed phenotypical and morphologic features undetectable in DCs generated in the presence of interleukin 4 (IL-4) and GM-CSF, such as expression of CD83 and CD25 and the presence of CD44+, highly polarized, thin, and long dendrites. IFN-DCs markedly migrated in response to β-chemokines (especially MIP-1β) and expressed the Th-1 chemokine IP-10. Notably, IFN-DCs showed an up-regulation of CCR7 as well as of its natural ligand MIP-3β, characteristics typical of mature DCs. Of interest, IFN-DCs exhibited a marked chemotactic response to MIP-3β in vitro and strong migratory behavior in severe combined immunodeficient (SCID) mice. In SCID mice reconstituted with human peripheral blood leukocytes, IFN-DCs induced a potent primary human antibody response and IFN-γ production, indicative of a Th-1 immune response. These results define the highly specialized maturation state of IFN-DCs and point out the existence of a “natural alliance” between type I IFN and monocyte/DC development, instrumental for ensuring an efficient connection between innate and adaptive immunity.

Type I interferons (IFNs) are cytokines exhibiting antiviral and antitumor effects, including multiple activities on immune cells. However, the importance of these cytokines in the early events leading to the generation of an immune response is still unclear. Here, we have investigated the effects of type I IFNs on freshly isolated granulocyte/macrophage colony-stimulating factor (GM-CSF)–treated human monocytes in terms of dendritic cell (DC) differentiation and activity in vitro and in severe combined immunodeficiency mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID) mice. Type I IFNs induced a surprisingly rapid maturation of monocytes into short-lived tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)–expressing DCs endowed with potent functional activities, superior with respect to the interleukin (IL)-4/GM-CSF treatment, as shown by FACS® analyses, mixed leukocyte reaction assays with allogeneic PBLs, and lymphocyte proliferation responses to HIV-1–pulsed autologous DCs. Type I IFN induced IL-15 production and strongly promoted a T helper cell type 1 response. Notably, injection of IFN-treated HIV-1–pulsed DCs in SCID mice reconstituted with autologous PBLs resulted in the generation of a potent primary immune response, as evaluated by the detection of human antibodies to various HIV-1 antigens. These results provide a rationale for using type I IFNs as vaccine adjuvants and support the concept that a natural alliance between these cytokines and monocytes/DCs represents an important early mechanism for connecting innate and adaptive immunity.

The presence of HIV-1 in the intestinal mucosa of AIDS patients has been reported and human intestinal lamina propria lymphocytes (LPL) have been proposed as important targets for HIV-1 infection. However, little information is available concerning the permissiveness of human intestinal CD4+ T lymphocytes to HIV-1 infection. Here, we show that human LPL, in contrast to autologous peripheral blood lymphocytes (PBL), are permissive to both X4 T-tropic and R5 M-tropic strains of HIV-1, as well as to clinical isolates, in the absence of exogenous stimuli. Flow cytometry showed that the vast majority of T LPL were CD45RO+ and CD69+, and that CD4+ T LPL highly expressed CC chemokine receptor 5 (CCR5) as compared to PBL, while CX chemokine receptor 4 was equally expressed on LPL and PBL. Exogenous RANTES and macrophage inflammatory protein-1alpha (natural CCR5 ligands) virtually abolished the entry of the R5 M-tropic strain HIV-1 into human LPL. Thus, we infer that human intestinal CD4+ T lymphocytes are naturally susceptible to HIV-1 infection, due to their physiological state of activation and to marked expression of HIV-1 coreceptors, independently of the route of primary (either mucosal or parental) infection and the shifts of the virus phenotype occurring during the course of AIDS.

In this article, we show that passage in SCID mice rendered a human CD4 + T-cell line (CEM cells) highly susceptible to infection by macrophage-tropic (M-tropic) strains and primary clinical isolates of human immunodeficiency virus type 1 (HIV-1). This in vivo-acquired permissiveness of CEM cells was associated with the induction of a CD45RO + phenotype as well as of some β-chemokine receptors. Regulated upon activation, normal T-cell expressed and secreted chemokine entirely inhibited the ability of M-tropic HIV-1 strains to infect these cells. These findings may lead to new approaches in investigating in vivo the capacity of different HIV strains to exploit chemokine receptors in relation to the dynamics of the activation and/or differentiation state of human CD4 + T cells.

Intrinsic resistance to cytotoxic drugs has been a main issue in cancer therapy for decades. Microenvironmental acidity is a simple while highly efficient mechanism of chemoresistance, exploited through impairment of drug delivery. The latter is achieved by extracellular protonation and/or sequestration into acidic vesicles. This study investigates the importance of extracellular acidosis and nanovesicle (exosome) release in the resistance of human tumour cell to cisplatin (CisPt); in parallel to proton pump inhibitors (PPI) ability of interfering with these tumour cell features. The results showed that CisPt uptake by human tumour cells was markedly impaired by low pH conditions. Moreover, exosomes purified from supernatants of these cell cultures contained various amounts of CisPt, which correlated to the pH conditions of the culture medium. HPLC-Q-ICP-MS analysis revealed that exosome purified from tumour cell culture supernatants contained CisPt in its native form. PPI pre-treatment increased cellular uptake of CisPt, as compared to untreated cells, in an acidic-depend manner. Furthermore, it induced a clear inhibition of exosome release by tumour cells. Human tumours obtained from xenografts pretreated with PPI contained more CisPt as compared to tumours from xenografts treated with CisPt alone. Further analysis showed that in vivo PPI treatment induced a clear reduction in the plasmatic levels of tumour-derived exosomes which also contained lower level of CisPt. Altogether, these findings point to the identification of a double mechanism that human malignant melanoma use in resisting to a dreadful cellular poison such as cisplatin. This framework of resistance includes both low pH-dependent extracellular sequestration and an exosome-mediated elimination. Both mechanisms are markedly impaired by proton pump inhibition, leading to an increased CisPt-dependent cytotoxicity.

The state of activation of the immune system may be an important factor which renders a host more receptive to human immunodeficiency virus (HIV) and more vulnerable to its effects. To explore this issue with a practical in vivo model, we developed a modified protocol of HIV infection in hu-PBL-SCID mice. First, we assessed the time course of activation of human peripheral blood lymphocytes (hu-PBL) in the peritoneal cavity of SCID mice. At 2 to 24 h after the intraperitoneal injection into SCID mice, there was a clear-cut increase in the percentage of hu-PBL expressing early activation markers (CD69), concomitant with the release of soluble intercellular adhesion molecule-1 (sICAM-1) and the soluble interleukin-2 receptor (sIL-2R) and with the accumulation of mRNAs for a number of human cytokines. At 2 weeks, virtually all of the hu-PBL expressed the memory phenotype (CD45RO) and HLA-DR antigens as well. Cells collected from the SCID mouse peritoneum at 2 and 24 h after transplantation were fully susceptible to in vitro infection with HIV type 1 (HIV-1) in the absence of either IL-2 or mitogens. The injection of HIV into hu-PBL-SCID mice at 2 h after reconstitution resulted in a generalized and productive HIV infection of the xenochimeras. This early HIV-1 infection resulted in a dramatic depletion of human CD4+ cells and in decreased levels of sICAM-1 (in the peritoneal lavage fluid) as well as of sIL-2R and immunoglobulins M and A (in the serum). Enzyme-linked immunosorbent assay and/or reverse transcriptase PCR analysis showed higher levels of IL-4, IL-5, and IL-10 in the HIV-infected animals than in control hu-PBL-SCID mice, while gamma interferon levels in the two groups were comparable. When we compared the current model of HIV-1 infection at 2 weeks after the intraperitoneal injection of the hu-PBL in the SCID mice with the model described here, we found that the majority of immune dysfunctions induced in the 2-h infection of the xenochimeras are not inducible in the 2-week infection. This supports the concept that the state of activation of human cells at the moment of the in vivo infection with HIV-1 is a crucial factor in determining the immune derangement observed in AIDS patients. These results show that some immunological dysfunctions induced by HIV infection in AIDS patients can be mimicked in this xenochimeric model. Thus, the hu-PBL-SCID mouse model may be useful in exploring, in vivo, the relevance of hu-PBL activation and differentiation in HIV-1 infection and for testing therapeutic intervention directed towards either the virus or the immune system.

Nanomedicine aims to exploit the improved and often novel physical, chemical, and biological properties of materials at the nanometric scale, possibly with the highest level of biomimetism, an approach that simulates what occurs in nature. Although extracellularly released vesicles include both microvesicles (MVs) and exosomes, only exosomes have the size that may be considered suitable for potential use in nanomedicine. In fact, recent reports have shown that exosomes are able to interact with target cells within an organ or at a distance using different mechanisms. Much is yet to be understood about exosomes, and currently, we are looking at the visible top of an iceberg, with most of what we have to understand on these nanovesicles still under the sea. In fact, we know that exosomes released by normal cells always trigger positive effects, whereas those released by cells in pathological condition, such as tumor or infected cells, may induce undesired, dangerous, and mostly unknown effects, but we cannot exclude the possibility that exosomes may also be detrimental for the body in normal conditions. However, whether we consider extracellular vesicles as a whole, thus including MVs, it appears that even in normal conditions, extracellular vesicles may lead to unwanted effects, depending on gender and age. This review aims to critically emphasize existing data in the literature that support the possible roles of exosomes in both diagnostic and therapeutic scopes.

Metastatic melanoma is associated with poor prognosis and still limited therapeutic options. An innovative treatment approach for this disease is represented by targeting acidosis, a feature characterizing tumor microenvironment and playing an important role in cancer malignancy. Proton pump inhibitors (PPI), such as esomeprazole (ESOM) are prodrugs functionally activated by acidic environment, fostering pH neutralization by inhibiting proton extrusion. We used human melanoma cell lines and xeno-transplated SCID mice to provide preclinical evidence of ESOM antineoplastic activity. Human melanoma cell lines, characterized by different mutation and signaling profiles, were treated with ESOM in different pH conditions and evaluated for proliferation, viability and cell death. SCID mice engrafted with human melanoma were used to study ESOM administration effects on tumor growth and tumor pH by magnetic resonance spectroscopy (MRS). ESOM inhibited proliferation of melanoma cells in vitro and induced a cytotoxicity strongly boosted by low pH culture conditions. ESOM-induced tumor cell death occurred via rapid intracellular acidification and activation of several caspases. Inhibition of caspases activity by pan-caspase inhibitor z-vad-fmk completely abrogated the ESOM-induced cell death. ESOM administration (2.5 mg kg-1) to SCID mice engrafted with human melanoma reduced tumor growth, consistent with decrease of proliferating cells and clear reduction of pH gradients in tumor tissue. Moreover, systemic ESOM administration dramatically increased survival of human melanoma-bearing animals, in absence of any relevant toxicity. These data show preclinical evidence supporting the use of PPI as novel therapeutic strategy for melanoma, providing the proof of concept that PPI target human melanoma modifying tumor pH gradients. © 2009 UICC.

Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.

Tumour cannibalism is a characteristic of malignancy and metastatic behaviour. This atypical phagocytic activity is a crucial survival option for tumours in conditions of low nutrient supply, and has some similarities to the phagocytic activity of unicellular microorganisms. In fact, Dictyostelium discoideum has been used widely as a model to study phagocytosis. Recently, phg1A has been described as a protein that is primarily involved in the phagocytic process of this microorganism. The closest human homologue to phg1A is transmembrane 9 superfamily protein member 4 (TM9SF4). Here, we report that TM9SF4 is highly expressed in human malignant melanoma cells deriving from metastatic lesions, whereas it is undetectable in healthy human tissues and cells. TM9SF4 is predominantly expressed in acidic vesicles of melanoma cells, in which it co-localizes with the early endosome antigens Rab5 and early endosome antigen 1. TM9SF4 silencing induced marked inhibition of cannibal activity, which is consistent with a derangement of intracellular pH gradients, with alkalinization of acidic vesicles and acidification of the cell cytosol. We propose TM9SF4 as a new marker of malignancy, representing a potential new target for anti-tumour strategies with a specific role in tumour cannibalism and in the establishment of a metastatic phenotype.

The membrane cytoskeleton cross-linker, ezrin, has recently been depicted as a key regulator in the progression and metastasis of several pediatric tumors. Less defined appears the role of ezrin in human adult tumors, especially melanoma. We therefore addressed ezrin involvement in the metastatic phenotype of human adult metastatic melanoma cells. Our results show that cells resected from melanoma metastatic lesions of patients, display marked metastatic spreading capacity in SCID mice organs. Stable transfection of human melanoma cells with an ezrin deletion mutant comprising only 146 N-terminal aminoacids led to the abolishment of metastatic dissemination. In vitro experiments revealed ezrin direct molecular interactions with molecules related to metastatic functions such as CD44, merlin and Lamp-1, consistent with its participation to the formation of phagocitic vacuoles, vesicular sorting and migration capacities of melanoma cells. Moreover, the ezrin fragment capable of binding to CD44 was shorter than that previously reported, and transfection with the ezrin deletion mutant abrogated plasma membrane Lamp-1 recruitment. This study highlights key involvement of ezrin in a complex machinery, which allows metastatic cancer cells to migrate, invade and survive in very unfavorable conditions. Our in vivo and in vitro data reveal that ezrin is the hub of the metastatic behavior also in human adult tumors.

BACKGROUND: Metastatic melanoma is an untreatable cancer lacking reliable and non-invasive markers of disease progression. Exosomes are small vesicles secreted by normal as well as tumor cells. Human tumor-derived exosomes are involved in malignant progression and we evaluated the presence of exosomes in plasma of melanoma patients as a potential tool for cancer screening and follow-up. METHODOLOGY/PRINCIPAL FINDINGS: We designed an in-house sandwich ELISA (Exotest) to capture and quantify exosomes in plasma based on expression of housekeeping proteins (CD63 and Rab-5b) and a tumor-associated marker (caveolin-1). Western blot and flow cytometry analysis of exosomes were used to confirm the Exotest-based findings. The Exotest allowed sensitive detection and quantification of exosomes purified from human tumor cell culture supernatants and plasma from SCID mice engrafted with human melanoma. Plasma levels of exosomes in melanoma-engrafted SCID mice correlated to tumor size. We evaluated the levels of plasma exosomes expressing CD63 and caveolin-1 in melanoma patients (n = 90) and healthy donors (n = 58). Consistently, plasma exosomes expressing CD63 (504+/-315) or caveolin-1 (619+/-310) were significantly increased in melanoma patients as compared to healthy donors (223+/-125 and 228+/-102, respectively). While the Exotest for CD63+ plasma exosomes had limited sensitivity (43%) the Exotest for detection of caveolin-1+ plasma exosomes showed a higher sensitivity (68%). Moreover, caveolin-1+ plasma exosomes were significantly increased with respect to CD63+ exosomes in the patients group. CONCLUSIONS/SIGNIFICANCE: We describe a new non-invasive assay allowing detection and quantification of human exosomes in plasma of melanoma patients. Our results suggest that the Exotest for detection of plasma exosomes carrying tumor-associated antigens may represent a novel tool for clinical management of cancer patients.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) and thus play a pivotal role in induction of the immune response. Recent studies in both human and mouse models have shown that type I IFN, cytokines originally characterized for their antiviral activity and exerting multiple biologic effects, efficiently promote the differentiation and activation of DCs. These observations, together with the findings that DCs can express biologically relevant levels of type I interferon (IFN) and, in particular, that high amounts of these cytokines are released by specialized DC precursors (i.e., plasmacytoid DCs) in response to viral infections, strongly suggest the existence of a natural alliance between type I IFN and DCs, which is instrumental in ensuring an efficient immune response to both infectious agents and tumors. Further recent knowledge on the interactions between type I IFN and DCs emphasizes the importance of these cytokines in linking innate and adaptive immunity and may lead to new perspectives in their use as vaccine adjuvants as well as in strategies for the development of DC-based vaccines.

CD95 (APO-1/Fas) is a member of the tumor necrosis factor receptor family, which can trigger apoptosis in a variety of cell types. However, little is known of the mechanisms underlying cell susceptibility to CD95-mediated apoptosis. Here we show that human T cells that are susceptible to CD95-mediated apoptosis, exhibit a constitutive polarized morphology, and that CD95 colocalizes with ezrin at the site of cellular polarization. In fact, CD95 co-immunoprecipitates with ezrin exclusively in lymphoblastoid CD4(+) T cells and primary long-term activated T lymphocytes, which are prone to CD95-mediated apoptosis, but not in short-term activated T lymphocytes, which are refractory to the same stimuli, even expressing equal levels of CD95 on the cell membrane. Pre-treatment with ezrin antisense oligonucleotides specifically protected from the CD95-mediated apoptosis. Moreover, we show that the actin cytoskeleton integrity is essential for this function. These findings strongly suggest that the CD95 cell membrane polarization, through an ezrin-mediated association with the actin cytoskeleton, is a key intracellular mechanism in rendering human T lymphocytes susceptible to the CD95-mediated apoptosis.

Although several studies are available on the in vitro inhibitory activities of type I interferon (IFN) on HIV-1 replication, the role of these cytokines in the pathogenesis of AIDS is still matter of conjecture. Both beneficial and adverse effects have been envisaged and considered as a possible rationale for the development of either IFN or anti-IFN therapies in HIV-1-infected patients. In the present study, we have evaluated the efficacy of human type I IFN on HIV-1 infection and virus-induced depletion of human CD4 T cells in two models established in SCID mice. In SCID mice transplanted with human U937 cells (U937-SCID mouse model), continuous treatment with type I consensus IFN (CIFN) resulted in a total suppression of HIV-1 infection. This inhibitory effect was superior to that obtained after AZT treatments. Results from an ensemble of experiments in SCID mice transplanted with either control or genetically modified human U937 cells transduced with a Tat-inducible IFN-α gene (LTR-IFN-A2 U937) indicated that low levels of IFN-α, produced locally as a result of virus infection, were extremely effective in inhibiting acute HIV infection and virus replication. Of interest, LTR-IFN-A2 U937 cells conferred a strong anti-HIV-1 protection to coinjected bystander U937 cells. Notably, experiments with SCID mice reconstituted with human PBL (hu-PBL-SCID mouse model) showed that treatment with CIFN inhibited HIV-1 replication more effectively than AZT treatment. Remarkably, treatment with CIFN resulted in a clear-cut protection from the virus-induced depletion of human CD4 T cells, which was also associated with the generation of an antibody response toward HIV-1 antigens in 50% of the virus-injected xenografts. These results suggest that type I IFN efficiently preserves human CD4 + cells from virus-induced damage in hu-PBL-SCID mice, not only by inducing an antiviral state in target cells but also by stimulating anti-HIV-1 human immune responses in vivo.

Swiss mice were injected intraperitoneally with uninfected or human immunodeficiency virus type 1 (HIV-1) infected human U937 cells. At 6 days, no residual human cells were detected in mouse tissues as determined by PCR analysis of DNAs from injected mice using primers and probes for the human HLA-DQ alpha gene. At 6 to 12 months, approximately 60% of the HIV-1-infected mice had antibodies to HIV-1 gp 120 and gp41 proteins. Fifteen percent of the animals showed evidence of HIV-1 infection as determined by PCR analyses of DNA from peripheral blood leukocytes and by in situ hybridization for detection of HIV-1 mRNA in peritoneal cells. In this set of experiments, spleen cells from mice sacrificed at different times after injection were cultured for 48 h in the presence or absence of mitogens [i.e.: concanavalin (Con A) or anti-CD3 antibody] and then tested for lymphocyte proliferation. At 10 to 12 months, splenocytes from approximately 80% of Swiss mice injected with HIV-1-infected U937 cells exhibited a marked defect in their proliferative response to Con A or anti-CD3 antibody as compared with spleen cells from both uninjected or U937 cell-injected mice. Similar results were obtained at 12 months in C3H/HeJ mice. Non-responding spleen cells from HIV-1-injected Swiss mice did not proliferate in response to anti-CD3 antibody even in the presence of co-stimulatory molecules such as phorbol myristate acetate or anti-CD28 antibody. Splenocytes from these mice also exhibited an impaired capacity to produce interferon-gamma and interleukin-4 after mitogen stimulation. No T cell defects were observed in control-injected mice. Immunofluorescence analyses revealed a significant decrease in the percentage of both CD4+ and CD8+ spleen cells in HIV-1-injected mice. These data indicate that immunocompetent mice can be used to investigate some HIV-1-related immune dysfunctions in vivo.

IL-7 promotes survival of resting T lymphocytes and induces T cell proliferation in lymphopenic conditions. As elevated IL-7 levels occur in HIV-infected individuals in addition to high Fas expression on T cells and increased sensitivity to Fas-induced apoptosis, we analyzed whether IL-7 has a regulatory role in Fas-mediated T cell apoptosis. We show that IL-7 up-regulates Fas expression on naive and memory T cells through a mechanism that involves translocation of Fas molecules from intracellular compartments to the cell membrane. IL-7 induced the association of Fas with the cytoskeletal component ezrin and a polarized Fas expression on the cell surface. The potential role of IL-7 in Fas up-regulation in vivo was verified in IL-7-treated macaques and in HIV-infected or chemotherapy treated patients by the correlation between serum IL-7 levels and Fas expression on T cells. IL-7 treatment primed T cells for Fas-induced apoptosis in vitro and serum IL-7 levels correlated with the sensitivity of T cells to Fas-induced apoptosis in HIV-infected individuals. Our data suggest an important role for IL-7 in Fas-mediated regulation of T cell homeostasis. Elevated IL-7 levels associated with lymphopenic conditions, including HIV-infection, might participate in the increased sensitivity of T cells for activation-induced apoptosis.

We have recently shown that a human CD4+ T cell line (CEM-SS) acquires the permissiveness to M-tropic strains and primary isolates of HIV-1 after transplantation into SCID mice. This permissiveness was associated with the acquisition of a memory (CD45RO+) phenotype as well as of a functional CCR5 coreceptor. In this study, we have used this model for invest-igating in vivo the relationships between HIV-1 infection, apoptosis and T cell differentiation. When an in vivo HIV-1 infection was performed, the CEM cell tumors grew to a lower extent than the uninfected controls. CEM cells explanted from uninfected SCID mice (ex vivo CEM) underwent a significant level of spontaneous apoptosis and proved to be CD45RO+, Fas+ and Fas-L+, while Bcl-2 expression was significantly reduced as compared to the parental cells. Acute HIV-1 infection markedly increased apoptosis of uninfected ex vivo CEM cells, through a Fas/Fas-L-mediated autocrine suicide/fratricide, while parental cells did not undergo apoptosis following viral infection. The susceptibility to apoptosis of ex vivo CEM cells infected with the NSI strain of HIV-1, was progressively lost during culture, in parallel with the loss of Fas-L and marked changes in the Bcl-2 cellular distribution. On the whole, these results are strongly reminiscent of a series of events possibly occurring during HIV-1 infection. After an initial depletion of bystander CD4+ memory T cells during acute infection, latently or chronically infected CD4+ T lymphocytes are progressively selected and are protected against spontaneous apoptosis through the development of an efficient survival program. Studies with human cells passaged into SCID mice may offer new opportunities for an in vivo investigation of the mechanisms involved in HIV-1 infection and CD4+ T cell depletion.