Role of mesenchymal stromal cell-derived extracellular vesicles in tumour microenvironment.

Stromal cells, deriving from mesenchymal stromal cells (MSCs), are crucial component of tumour microenvironment and represent key regulators of tumour processes. MSCs can be recruited to the tumour environment and interact with many cellular elements, thus influencing tumour biology. Cell-to-cell communication is in part mediated by the release of extracellular vesicle (EVs). EVs can induce significant molecular changes in recipient cells, delivering bioactive molecules. In this review, we describe the MSC-derived EVs content and discuss their role in different processes related to cancer biology. Furthermore, we summarize chemical or biological EVs modifications aiming to develop more efficient antitumor therapies.

Ketamine enhances structural plasticity in mouse mesencephalic and human iPSC-derived dopaminergic neurons via AMPAR-driven BDNF and mTOR signaling.

Among neurobiological mechanisms underlying antidepressant properties of ketamine, structural remodeling of prefrontal and hippocampal neurons has been proposed as critical. The suggested mechanism involves downstream activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which trigger mammalian target of rapamycin (mTOR)-dependent structural plasticity via brain-derived neurotrophic factor (BDNF) and protein neo-synthesis. We evaluated whether ketamine elicits similar molecular events in dopaminergic (DA) neurons, known to be affected in mood disorders, using a novel, translational strategy that involved mouse mesencephalic and human induced pluripotent stem cells-derived DA neurons. Sixty minutes exposure to ketamine elicited concentration-dependent increases of dendritic arborization and soma size in both mouse and human cultures as measured 72 hours after application. These structural effects were blocked by mTOR complex/signaling inhibitors like rapamycin. Direct evidence of mTOR activation by ketamine was revealed by its induction of p70S6 kinase. All effects of ketamine were abolished by AMPA receptor antagonists and mimicked by the AMPA-positive allosteric modulator CX614. Inhibition of BDNF signaling prevented induction of structural plasticity by ketamine or CX614. Furthermore, the actions of ketamine required functionally intact dopamine D3 receptors (D3R), as its effects were abolished by selective D3R antagonists and absent in D3R knockout preparations. Finally, the ketamine metabolite (2R,6R)-hydroxynorketamine mimicked ketamine effects at sub-micromolar concentrations. These data indicate that ketamine elicits structural plasticity by recruitment of AMPAR, mTOR and BDNF signaling in both mouse mesencephalic and human induced pluripotent stem cells-derived DA neurons. These observations are of likely relevance to the influence of ketamine upon mood and its other functional actions in vivo.

Geochemical behavior of an acid drainage system: the case of the Amarillo River, Famatina (La Rioja, Argentina).

The Amarillo River (Famatina range, Argentina, ~29° S and ~67° W) is unusual because acid mine drainage (AMD) is superimposed on the previously existing acid rock drainage (ARD) scenario, as a Holocene paleolake sedimentary sequence shows. In a markedly oxidizing environment, its water is currently ferrous and of the sulfate-magnesium type with high electrical conductivity (>10 mS cm-1 in uppermost catchments). At the time of sampling, the interaction of the mineralized zone with the remnants of mining labors determined an increase in some elements (e.g., Cu ~3 to ~45 mg L-1; As ~0.2 to ~0.5 mg L-1). Dissolved concentrations were controlled by pH, decreasing significantly by precipitation of neoformed minerals (jarosite and schwertmannite) and subsequent metal sorption (~700 mg kg-1 As, 320 mg kg-1 Zn). Dilution also played a significant role (i.e., by the mixing with circumneutral waters which reduces the dissolved concentration and also enhances mineral precipitation). Downstream, most metals exhibited a significant attenuation (As 100 %, Fe 100 %, Zn 99 %). PHREEQC-calculated saturation indices (SI) indicated that Fe-bearing minerals, especially schwertmannite, were supersaturated throughout the basin. All positive SI increased through the input of circumneutral water. PHREEQC inverse geochemical models showed throughout the upper and middle basin, that about 1.5 mmol L-1 of Fe-bearing minerals were precipitated. The modeling exercise of mixing different waters yielded results with a >99 % of correlation between observed and modeled data.

Human cartilage fragments in a composite scaffold for single-stage cartilage repair: an in vitro study of the chondrocyte migration and the influence of TGF-ß1 and G-CSF.

PURPOSE: Minced chondral fragments are becoming popular as a source of cells for cartilage repair, as a growing interest is developing towards one-stage procedures to treat cartilage lesions. The purpose of this study is to (A) compare cell outgrowth from cartilage fragments of adult and young donors using two different types of scaffolds and (B) evaluate the influence of transforming-growth-factor-ß1 (TGF-ß1) and granulocyte colony-stimulating factor (G-CSF) on chondrocyte behaviour. METHODS: In part (A) cartilage fragments from adult and young donors were either loaded onto an HA-derivative injectable paste scaffold or onto an HA-derivative membrane scaffold. Construct sections were then examined for cell counting after 1, 2 and 3 months. In part (B) only membrane scaffolds were prepared using cartilage fragments from young donors. Constructs were cultured either in standard growth medium or in the presence of specific growth factors, such as TGF-ß1 or G-CSF or TGF-ß1 + G-CSF. After 1 month, construct sections were examined for cell counting. Expression of chondrocyte markers (SOX9, CD151, CD49c) and proliferative markers (ß-catenin, PCNA) was assessed using immunofluorescence techniques, both in unstimulated construct sections and in cells from unstimulated and stimulated construct cultures. RESULTS: Part (A): histological analysis showed age-dependent and time-dependent chondrocyte migration. A significant difference (p < 0.05) was observed between young and older donors at the same time point. No difference was detected between the two types of scaffolds within the same group at the same time point. Part (B): after 1 month, the number of migrating cells/area significantly increased due to exposure to TGF-ß1 and/or G-CSF (p < 0.05). Immunofluorescence revealed that outgrowing cells from unstimulated scaffold sections were positive for SOX9, CD151, CD49c and G-CSF receptor. Immunofluorescence of cells from construct cultures showed an increase in ß-catenin in all stimulated groups and an increased PCNA expression in G-CSF-exposed cultures (p < 0.05). CONCLUSION: Outgrowing cells may represent a subset of chondrocytes undergoing a phenotypic shift towards a proliferative state. TGF-ß1, and to a greater extent G-CSF, may accelerate this outgrowth. The clinical relevance of this study may involve a potential future clinical application of scaffolds preloaded with growth factors as an additional coating for chondral fragments. Indeed, a controlled delivery of G-CSF, widely employed in various clinical settings, might improve the repair process driven by minced human cartilage fragments during one-stage cartilage repair.

Treatment of medial and posteromedial knee instability: indications, techniques, and review of the results.

Injuries of the posteromedial corner of the knee are relatively common. These can be isolated or combined with other ligament lesions. In some cases the treatment of postero-medial corner injuries is controversial. After a brief description of the anatomy and biomechanics of the medial side of the knee, this paper reviews the indications for isolated and multiligamentous medial/posteromedial corner injuries both in the acute and in the chronic setting. In addition, the most common surgical techniques for repair and reconstruction are described in addition to outcomes based upon a review of the literature.

Mouse dendritic cells express the P2X7 purinergic receptor: characterization and possible participation in antigen presentation.

Immune cells express P2 purinoceptors of the P2Y and P2X subtypes. In the present work, we show that three dendritic cell (DC) lines, D2SC/1, CB1, and FSDC, representative of immature DCs, express the P2X7 (formerly P2Z) receptor, as judged from RT-PCR amplification, reactivity to a specific antiserum, and pharmacological and functional evidence. Receptor expression is higher in FSDC cells, a cell line that is functionally more mature than D2SC/1 and CB1. From the wild-type DC population, we selected cell clones lacking the P2X7R (P2X7less). We also used a P2XR blocker, oxidized ATP, to irreversibly inhibit the P2X7R. Ability of P2X7less FSDCs or of oxidized ATP-inhibited FSDCs to stimulate Ag-specific TH lymphocytes was severely decreased although Ag endocytosis was minimally affected. During coculture with TH lymphocytes, wild-type FSDC secreted large amounts of IL-1beta. Release of this cytokine was reduced in P2X7less DCs. These data show that DCs express the P2X7 purinoceptor and suggest a correlation between P2X7R expression and Ag-presenting activity.

Endothelial cell integrin alpha5beta1 expression is modulated by cytokines and during migration in vitro.

Alterations in endothelial cell-extracellular matrix interactions are central to the process of angiogenesis. We have investigated the effect of wound-induced two-dimensional migration, basic fibroblast growth factor (bFGF), transforming growth factor-beta1 (TGF-beta1) and leukemia inhibitory factor (LIF) on expression of the alpha5beta1 integrin in endothelial cells. In multiple-wounded monolayers of bovine microvascular endothelial (BME) cells, an increase in mRNA and total protein for both alpha5 and beta1 subunits was observed, and this could be correlated with a reduction in cell density but not proliferation, both of which are induced following wounding. Although as previously reported, the alpha5 subunit was increased when cells were exposed to TGF-beta1 alone, co-addition of bFGF and TGF-beta1 resulted in a striking synergistic induction of alpha5, with no significant changes in the expression of beta1. In contrast, the alpha5 subunit was decreased by LIF in bovine aortic endothelial but not in BME cells. These findings suggest that quantitative alterations in alpha5 and beta1 integrin subunit expression modulate the adhesive and migratory properties of endothelial cells during angiogenesis.

Blockade of human P2X7 receptor function with a monoclonal antibody.

A monoclonal antibody (MoAb) specific for the human P2X7 receptor was generated in mice. As assessed by flow cytometry, the MoAb labeled human blood-derived macrophage cells natively expressing P2X7 receptors and cells transfected with human P2X7 but not other P2X receptor types. The MoAb was used to immunoprecipitate the human P2X7 receptor protein, and in immunohistochemical studies on human lymphoid tissue, P2X7 receptor labeling was observed within discrete areas of the marginal zone of human tonsil sections. The antibody also acted as a selective antagonist of human P2X7 receptors in several functional studies. Thus, whole cell currents, elicited by the brief application of 2',3'-(4-benzoyl)-benzoyl-ATP in cells expressing human P2X7, were reduced in amplitude by the presence of the MoAb. Furthermore, preincubation of human monocytic THP-1 cells with the MoAb antagonized the ability of P2X7 agonists to induce the release of interleukin-1beta.

Differential onset of expression of alpha 7 and beta 1D integrins during mouse heart and skeletal muscle development.

beta 1D is a recently identified isoform of the beta 1 integrin subunit selectively expressed in skeletal and cardiac muscles. In the present study we determined the temporal expression of beta 1D and its association with alpha subunits during mouse development. By immunohistochemistry and western blot analysis we demonstrated that beta 1D begins to be expressed in skeletal muscles of 17 days embryo (stage E17). Its level progressively increases reaching maximal values few days after birth and remaining high in adult mice. At earlier stages of development (E11-E17) the beta 1A isoform is expressed in skeletal muscle cells. After E17 beta 1A is downregulated and disappears from muscle fibers few days after birth. In cardiac muscle the regulation of the beta 1D expression is different: beta 1D and beta 1A are coexpressed in the heart of E11 embryo. Subsequently expression of beta 1A declines, while beta 1D increases until it becomes the unique beta 1 isoform in cardiomyocytes few days after birth. Previous studies (Belkin et al J. Cell Biol. 132: 211-226, 1996) demonstrated that beta 1D in adult mouse cardiomyocytes is exclusively associated with alpha 7B. Western blot analysis shows that alpha 7B starts to be expressed in the heart only at stage E17, while beta 1D is expressed already at E11 embryo, indicating that alpha subunits other than alpha 7 should associate with beta 1D in early developmental stages. To investigate this aspect, beta 1 associated alpha subunits were identified by western blotting from cardiomyocytes integrin complexes immunoprecipitated with alpha subunit specific antibodies. We found that, during cardiomyocyte development, beta 1D associates with several alpha subunits namely with alpha 5, alpha 6A and alpha 7B. In conclusion these data show that the expression of the beta 1D muscle specific integrin during development occurs much earlier in heart than in skeletal muscle and it can dimerize with different alpha subunits.

Myogenic conversion of NIH3T3 cells by exogenous MyoD family members: dissociation of terminal differentiation from myotube formation.

Myogenic regulatory factors (MRF) of the MyoD family regulate the skeletal muscle differentiation program. Non-muscle cells transfected with exogenous MRF either are converted to the myogenic lineage or fail to express the muscle phenotype, depending on the cell type analysed. We report here that MRF-induced myogenic conversion of NIH3T3 cells results in an incomplete reprogramming of these cells. Transfected cells withdrew from the cell cycle and underwent biochemical differentiation but, surprisingly, terminally differentiated myocytes absolutely failed to fuse into multinucleated myotubes. Analysis of muscle regulatory and structural gene expression failed to provide an explanation for the fusion defectiveness. However, myogenic derivatives of NIH3T3 cells were shown to be unable to accumulate the transcripts encoding muscle-specific isoforms of the integrin subunit beta1D and the transcription factor MEF2D1b2, that depend on muscle-specific alternative splicing. Our results suggest that the fusion into myotubes is under a distinct genetic control that might depend, at least partially, on differential splicing.

Tissue distribution of the P2X7 receptor.

The P2X7 receptor is a bifunctional molecule. The binding of ATP induces within milliseconds the opening of a channel selective for small cations, and within seconds a larger pore opens which allows permeation by molecules as large as propidium dyes (629 Da). In situ hybridization using a digoxigenin-labelled riboprobe, and immunohistochemistry using an antibody raised against a C-terminal peptide sequence, were used to determine the distribution of the P2X7 receptor mRNA and protein in rat and mouse tissues and cell lines. The brain of newborn rats showed a 6 kb RNA by Northern blotting, but this was not detectable in adult brain. By in situ hybridization and immunohistochemistry, there was heavy labelling of ependymal cells in both newborn and adult brain, but the brain parenchyma showed no labelling. However, P2X7 receptor-immunoreactive cells appeared in the penumbral region around an area of necrosis evoked by prior occlusion of the middle cerebral artery, suggesting expression of the receptor by activated microglia. NTW8 cells, a mouse microglial cell line, strongly expressed the P2X7 receptor mRNA and protein. The P2X7 receptor mRNA and protein were also observed in the majority of bone marrow cells, including those separately identified by their expression of other antigens as granulocytes, monocyte/macrophages and B lymphocytes. The expression of P2X7 receptor by brain macrophages rather than neurons would be consistent with a role in brain repair following inflammation, infarction or immune insult.

ATP-mediated cytotoxicity in microglial cells.

Microglial cells are known to express purinergic receptors for extracellular ATP of both the P2Y and P2X subtypes. Functional studies have shown that both primary mouse microglial cells and the N9 and N13 microglial cell lines express the pore-forming P2Z/P2X7 receptor. Here we identify the presence of this receptor in N9 and N13 cells with a specific polyclonal Ab and show that microglial cells expressing the P2Z/P2X7 receptor are exquisitively sensitive to ATP-mediated cytotoxicity while clones selected for the lack of this receptor are resistant. Transfection of HEK293 cells with P2X7 (but not P2X2) receptor cDNA confers susceptibility to ATP-mediated cytotoxicity. Morphological and biochemical analysis suggests that ATP-dependent cell death in microglial cells occurs by apoptosis. Finally, microglial cells release ATP via a non-lytic mechanism when activated by bacterial endotoxin, thus suggesting the operation of a purinergic autocrine/paracrine loop.

Spontaneous cell fusion in macrophage cultures expressing high levels of the P2Z/P2X7 receptor.

Mouse and human macrophages express a plasma membrane receptor for extracellular ATP named P2Z/P2X7. This molecule, recently cloned, is endowed with the intriguing property of forming an aqueous pore that allows transmembrane fluxes of hydrophylic molecules of molecular weight below 900. The physiological function of this receptor is unknown. In a previous study we reported experiments suggesting that the P2Z/P2X7 receptor is involved in the formation of macrophage-derived multinucleated giant cells (MGCs; Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, and F. Di Virgilio. 1995. J. Clin. Invest. 95:1207- 1216). We have selected several clones of mouse J774 macrophages that are characterized by either high or low expression of the P2Z/P2X7 receptor and named these clones P2Zhyper or P2Zhypo, respectively. P2Zhyper, but not P2Zhypo, cells grown to confluence in culture spontaneously fuse to form MGCs. As previously shown for human macrophages, fusion is inhibited by the P2Z/P2X7 blocker oxidized ATP. MGCs die shortly after fusion through a dramatic process of cytoplasmic sepimentation followed by fragmentation. These observations support our previous hypothesis that the P2Z/P2X7 receptor is involved in macrophage fusion.

The permeabilizing ATP receptor, P2X7. Cloning and expression of a human cDNA.

A cDNA was isolated from a human monocyte library that encodes the P2X7 receptor; the predicted protein is 80% identical to the rat receptor. Whole cell recordings were made from human embryonic kidney cells transfected with the human cDNA and from human macrophages. Brief applications (1-3 s) of ATP and 2', 3'-(4-benzoyl)-benzoyl-ATP elicited cation-selective currents. When compared with the rat P2X7 receptor, these effects required higher concentrations of agonists, were more potentiated by removal of extracellular magnesium ions, and reversed more rapidly on agonist removal. Longer applications of agonists permeabilized the cells, as evidenced by uptake of the propidium dye YO-PRO1, but this was less marked than for cells expressing the rat P2X7 receptor. Expression of chimeric molecules indicated that some of the differences between the rat and human receptor could be reversed by exchanging the intracellular C-terminal domain of the proteins.

Distinct alpha 7A beta 1 and alpha 7B beta 1 integrin expression patterns during mouse development: alpha 7A is restricted to skeletal muscle but alpha 7B is expressed in striated muscle, vasculature, and nervous system.

The laminin binding alpha 7 beta 1 integrin has been described as a major integrin in skeletal muscle. The RNA coding for the cytoplasmic domain of alpha 7 integrin undergoes alternative splicing to generate two major forms, denoted alpha 7A and alpha 7B. In the current paper, we have examined the developmental expression patterns of the alpha 7A and alpha 7B splice variants in the mouse. The alpha 7 integrin expression is compared to that of the nonintegrin laminin receptor dystroglycan and to that of laminin-alpha 1 and laminin-alpha 2 chains. Alpha 7A integrin was found by in situ hybridization to be specific to skeletal muscle. Antibodies specific for alpha 7B integrin and in situ hybridization revealed the presence of alpha 7 mRNA and alpha 7B protein in the E10 myotome and later in primary and secondary myotubes. In the heart, alpha 7B integrin was not detectable in the endocardium or myocardium during embryonic and fetal heart development. Northern blot analysis and immunohistochemistry revealed a postnatal induction of alpha 7B in the myocardium. In addition to striated muscle, alpha 7B integrin was localized to previously unreported nonmuscle locations such as a subset of vascular endothelia and restricted sites in the nervous system. Comparison of the alpha 7 integrin expression pattern with that of different laminin isoforms and dystroglycan revealed a coordinated temporal expression of dystroglycan, alpha 7 integrin, and laminin-alpha 2, but not laminin-alpha 1, in the forming skeletal muscle. We conclude that the alpha 7A and alpha 7B integrin variants are expressed in a developmentally regulated, tissue-specific pattern suggesting different functions for the two splice forms.

[Interstitial pulmonitis due to the cytomegalovirus in an immunocompetent adolescent: treatment with ganciclovir]. / Polmonite interstiziale da citomegalovirus in adolescente immunocompetente: trattamento con il Ganciclovir.

Interstitial pneumonia is the most common disease caused by infection from cytomegalovirus (CMV) in immunodepressed patients, whereas it is a rare complication in immunocompetent patients. With reference to the second group of patients, little literature has been produced as for the therapy to choose when symptoms are serious. We report the case of immunocompetent adolescent with CMV pneumonitis who responded dramatically to therapy with ganciclovir. For a week B. M., a 15-year-old girl, has been showing fever, cough and boring pain at her left thoracic base. When hospitalized, the girl was suffering and dyspneic, cardio-thoracic conditions were bad. Spleen and liver were palpable two fingers far form costal arch. Hematochemical tests showed an increase in phlogosis and transaminase value. Thoracic X-ray was negative, as well as cultures. Among the serological tests high response of anti-CMV IgM was remarkable. Virological blood test confirmed active CMV infection. On the fifth day, a thoracic radiography showed widespread interstitial infiltrates. Treatment with ganciclovir--i.v. 6 mg/kg/day, twice a day for twelve days--has been then adopted. After two day treatment, the girls was apyretic and eupneic. After ten day treatment, thoracic radiography was negative and a great decrease in CMV antigenic response was given by blood tests. No side effect were observed. According to our experience we can say that treatment with ganciclovir may positively shorten the course of pneumonia caused by CMV in immunocompetent patients.

Cloning OF P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels.

Two new P2X receptor cDNAs (P2X5 and P2X6) were isolated and expressed. All six proteins are 36-48 percent identical and seem to have two transmembrane segments with a large extracellular loop. Functionally, P2X5 and P2X6 receptors most resemble P2X2 and P2X4; they desensitize only slowly and do not respond to alpha beta methylene-ATP. P2X6 receptors, like P2X4, receptors, are not blocked by the antagonists suramin and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid. P2X6 and P2X5 receptors express at lower levels than P2X1-P2X4 receptors do, perhaps indicating that they do not normally form homomultimeric channels. P2X6 and P2X4 are the receptors expressed most heavily in brain, where their RNAs have a widespread and extensively overlapping distribution. The spinal cord expresses all receptors except P2X3. P2X2, P2X4, and P2X6, are the most abundant in the dorsal horn. Sensory neurons of the trigeminal, dorsal root, and nodose ganglia express all six RNAs; P2X3 is found only there. The functional properties and tissue distribution of these six P2X receptors indicate new roles for ATP-gated ion channels.

P2X1 receptor activation in HL60 cells.

Recent cloning of the human P2X1 receptor revealed high levels of its messenger RNA in differentiated promyelocytes (HL60 cells). We found expression of P2X1 receptor protein in HL60 cells by radioligand binding, by immunohistochemistry, using a receptor specific antibody, and by electrophysiology. The currents elicited by adenosine triphosphate (ATP) had the expected properties of P2X1 receptors (rapid desensitization, mimicked by alpha,beta-methylene-ATP). However, these currents were only observed in cells that were pretreated with apyrase, which destroys extracellular ATP, or with suramin, a P2X receptor antagonist. This implies that HL60 cells release ATP, which chronically desensitizes the receptor. ATP release was detected by direct measurement, using the luciferin-luciferase assay. It is concluded that functional P2X1 receptors are present in the membrane of differentiated HL60 cells.