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Immunoglobulin (Ig) isotype diversification by class switch recombination (CSR) is an essential process for mounting a protective humoral immune response. Ig CSR deficiencies in humans can result from an intrinsic B cell defect; however, most of these deficiencies are still molecularly undefined and diagnosed as common variable immunodeficiency (CVID). Here, we show that extracellular adenosine critically contributes to CSR in human naive and IgM memory B cells. In these cells, coordinate stimulation of B cell receptor and toll-like receptors results in the release of ATP stored in Ca2+-sensitive secretory vesicles. Plasma membrane ectonucleoside triphosphate diphosphohydrolase 1 CD39 and ecto-5′-nucleotidase CD73 hydrolyze ATP to adenosine, which induces CSR in B cells in an autonomous fashion. Notably, CVID patients with impaired class-switched antibody responses are selectively deficient in CD73 expression in B cells, suggesting that CD73-dependent adenosine generation contributes to the pathogenesis of this disease.

TCR signal strength instructs αβ versus γδ lineage decision in immature T cells. Increased signal strength of γδTCR with respect to pre-TCR results in induction of the γδ differentiation program. Extracellular ATP evokes physiological responses through purinergic P2 receptors expressed in the plasma membrane of virtually all cell types. In peripheral T cells, ATP released upon TCR stimulation enhances MAPK activation through P2X receptors. We investigated whether extracellular ATP and P2X receptors signaling tuned TCR signaling at the αβ/γδ lineage bifurcation checkpoint. We show that P2X7 expression was selectively increased in immature γδ+CD25+ cells. These cells were much more competent to release ATP than pre–TCR-expressing cells following TCR stimulation and Ca2+ influx. Genetic ablation as well as pharmacological antagonism of P2X7 resulted in impaired ERK phosphorylation, reduction of early growth response (Egr) transcripts induction, and diversion of γδTCR-expressing thymocytes toward the αβ lineage fate. The impairment of the ERK-Egr-inhibitor of differentiation 3 (Id3) signaling pathway in γδ cells from p2rx7−/− mice resulted in increased representation of the Id3-independent NK1.1-expressing γδ T cell subset in the periphery. Our results indicate that ATP release and P2X7 signaling upon γδTCR expression in immature thymocytes constitutes an important costimulus in T cell lineage choice through the ERK-Egr-Id3 signaling pathway and contributes to shaping the peripheral γδ T cell compartment.

Extracellular nucleotides regulate many cellular functions through activation of purinergic receptors in the plasma membrane. Here, we show that in hematopoietic stem cell (HSC), ATP is stored in vesicles and released in a calcium-sensitive manner. HSC expresses ATP responsive P2X receptors and in vitro pharmacological P2X antagonism restrained hematopoietic progenitors proliferation, but not myeloid differentiation. In mice suffering from chronic inflammation, HSCs were significantly expanded and their cycling activity was sensitive to treatment with the P2X antagonist periodate-oxidized 2,3-dialdehyde ATP. Our results indicate that ATP acts as an autocrine stimulus in regulating HSCs pool size.

The regulation of filopodia plays a crucial role during neuronal development and synaptogenesis. Axonal filopodia, which are known to originate presynaptic specializations, are regulated in response to neurotrophic factors. The structural components of filopodia are actin filaments, whose dynamics and organization are controlled by ensembles of actin-binding proteins. How neurotrophic factors regulate these latter proteins remains, however, poorly defined. Here, using a combination of mouse genetic, biochemical, and cell biological assays, we show that genetic removal of Eps8, an actin-binding and regulatory protein enriched in the growth cones and developing processes of neurons, significantly augments the number and density of vasodilator-stimulated phosphoprotein (VASP)-dependent axonal filopodia. The reintroduction of Eps8 wild type (WT), but not an Eps8 capping-defective mutant, into primary hippocampal neurons restored axonal filopodia to WT levels. We further show that the actin barbed-end capping activity of Eps8 is inhibited by brain-derived neurotrophic factor (BDNF) treatment through MAPK-dependent phosphorylation of Eps8 residues S624 and T628. Additionally, an Eps8 mutant, impaired in the MAPK target sites (S624A/T628A), displays increased association to actin-rich structures, is resistant to BDNF-mediated release from microfilaments, and inhibits BDNF-induced filopodia. The opposite is observed for a phosphomimetic Eps8 (S624E/T628E) mutant. Thus, collectively, our data identify Eps8 as a critical capping protein in the regulation of axonal filopodia and delineate a molecular pathway by which BDNF, through MAPK-dependent phosphorylation of Eps8, stimulates axonal filopodia formation, a process with crucial impacts on neuronal development and synapse formation.

Regulated expression of positive and negative regulatory factors controls the extent and duration of T cell adaptive immune response preserving the organism's integrity. Calreticulin (CRT) is a major Ca2+ buffering chaperone in the lumen of the endoplasmic reticulum. Here we investigated the impact of CRT deficiency on T cell function in immunodeficient mice reconstituted with fetal liver crt−/− hemopoietic progenitors. These chimeric mice displayed severe immunopathological traits, which correlated with a lower threshold of T cell receptor (TCR) activation and exaggerated peripheral T cell response to antigen with enhanced secretion of inflammatory cytokines. In crt−/− T cells TCR stimulation induced pulsatile cytosolic elevations of Ca2+ concentration and protracted accumulation of nuclear factor of activated T cells in the nucleus as well as sustained activation of the mitogen-activated protein kinase pathways. These observations support the hypothesis that CRT-dependent shaping of Ca2+ signaling critically contributes to the modulation of the T cell adaptive immune response.

The SNARE-dependent exocytosis of glutamate-containing vesicles in astrocytes is increasingly viewed as an important signal at the basis of the astrocyte-to-neurone communication system in the brain. Here we provide further insights into the molecular features and dynamics of vesicles in cultured astrocytes. We found that immunoisolated synaptobrevin2 vesicles are clear vesicles quite heterogenous in size and contain the vesicular glutamate transporter v-Glut-2. Moreover, they are immunopositive for synaptotagmin IV, for AMPA receptor subunits GluR2,3 and, to a lesser extent, for GluR1. We also provide direct evidence for the functional expression of v-Glut-2 in astrocytes and demonstrate that synaptobrevin2-positive vesicles can specifically take up (3H)L-glutamate via a bafilomycin-sensitive mechanism. Finally, by time lapse confocal microscopy, we show that a subpopulation of vesicles (tagged with a synaptobrevin2-EGFP chimera) is highly mobile and can fuse with the plasma membrane, preferentially at the level of the astrocyte processes, in a Ca2+-dependent manner. These latter observations, together with the evidence reported here for the expression of functional v-Glut-2 in synaptobrevin2-positive vesicles, provide a molecular basis for regulated exocytosis in astrocyte.

AMPA receptor ion channels are of paramount importance for postsynaptic excitation. Several reports demonstrate that AMPA receptors are present in the presynaptic compartment and point to a role of these receptors in the modulation of presynaptic function. We discuss here the possibility that not only ion influx through the receptor, but also biochemical cascades, activated by ligand binding and independent from ion flux, might contribute to AMPA mediated presynaptic modulation.

Over the past decade, evidence has accumulated indicating that, during development, the construction of synapses – the sites of communication between neurons – might rely on the utilization of preassembled sets of synaptic proteins, which have already accumulated in the axon and are highly mobile, before getting recruited to the sites of contact with the postsynaptic neuron. In this review, we discuss evidence from most recent publications pointing to the existence of active vesicle traffic and turnover in developing neurons, which lead to the construction of new synaptic sites.

Glutamine and alanine are important precursors for the synthesis of glutamate. Provided to neurons by neighboring astrocytes, these amino acids are internalized by classical system A amino acid carriers. In particular, System A transporter (SAT1) is a highly efficient glutamine transporter, whereas SAT2 exhibits broad specificity for neutral amino acids with a preference for alanine. We investigated the localization and the functional relevance of SAT1 and SAT2 in primary cultures of hippocampal neurons. Both carriers have been expressed since early developmental stages and are uniformly distributed throughout all neuronal processes. However, whereas SAT1 is present in axonal growth cones and can be detected at later developmental stages at the sites of synaptic contacts, SAT2 does not appear to be significantly expressed in these compartments. The non-metabolizable amino acid analogue alpha-(methylamino)-isobutyric acid, a competitive inhibitor of system A carriers, significantly reduced miniature excitatory postsynaptic current amplitude in neurons growing on top of astrocytes, being ineffective in pure neuronal cultures. alpha-(Methylamino)-isobutyric acid did not alter neuronal responsitivity to glutamate, thus excluding a postsynaptic effect. These data indicate that system A carriers are expressed with a different subcellular distribution in hippocampal neurons and play a crucial role in controlling the astrocyte-mediated supply of glutamatergic neurons with neurotransmitter precursors.

During development of neuronal circuits, presynaptic and postsynaptic functions are adjusted in concert, to optimize interneuronal signaling. We have investigated whether activation of glutamate receptors affects presynaptic function during synapse formation, when constitutive synaptic vesicle recycling is downregulated. Using primary cultures of hippocampal neurons as a model system, we have found that chronic exposure to both NMDA and non-NMDA glutamate receptor blockers during synaptogenesis produces an increase in miniature EPSC (mEPSC) frequency, with no significant changes in mEPSC amplitude or in the number of synapses. Enhanced synaptic vesicle recycling, selectively in glutamatergic nerve terminals, was confirmed by the increased uptake of antibodies directed against the lumenal domain of synaptotagmin. No increased uptake was detected in neuronal cultures grown in the chronic presence of TTX, speaking against an indirect effect caused by decreased electrical activity. Enhanced mEPSC frequency correlated with a reduction of synaptophysin-synaptobrevin–vesicle-associated membrane protein 2 (VAMP2) complexes detectable by immunoprecipitation. Intracellular perfusion with a peptide that inhibits the binding of synaptophysin to synaptobrevin–VAMP2 induced a remarkable increase of mEPSC frequency in control but not in glutamate receptor blocker-treated neurons. These findings suggest that activation of glutamate receptors plays a role in the downregulation of the basal rate of synaptic vesicle recycling that accompanies synapse formation. They also suggest that one of the mechanisms through which this downregulation is achieved is an increased interaction of synaptophysin with synaptobrevin–VAMP2.

Astrocytes possess different, efficient ways to generate complex changes in intracellular calcium concentrations, which allow them to communicate with each other and to interact with adjacent neuronal cells. Here we show that cultured hippocampal astrocytes coexpress the ectoenzyme CD38, directly involved in the metabolism of the calcium mobilizer cyclic ADP-ribose, and the NAD+ transporter connexin 43. We also demonstrate that hippocampal astrocytes can release NAD+ and respond to extracellular NAD+ or cyclic ADP-ribose with intracellular calcium increases, suggesting the existence of an autocrine cyclic ADP-ribose-mediated signalling. Cyclic ADP-ribose-induced calcium changes are in turn responsible for an increased glutamate and GABA release, this effect being completely inhibited by the cyclic ADP-ribose specific antagonist 8-NH2-cADPR. Furthermore, addition of NAD+ to astrocyte-neuron co-cultures results in a delayed intracellular calcium transient in neuronal cells, which is strongly but not completely inhibited by glutamate receptor blockers. These data indicate that an astrocyte-to-neuron calcium signalling can be triggered by the CD38/cADPR system, which, through the activation of intracellular calcium responses in astrocytes, is in turn responsible for the increased release of neuromodulators from glial cells.

Nitric oxide (NO) is synthesized by members of the NO synthase (NOS) family. Recently the existence of a mitochondrial NOS (mtNOS), its Ca(2+) dependence, and its relevance for mitochondrial bioenergetics was reported (Ghafourifar, P., and Richter, C. (1997) FEBS Lett. 418, 291-296; Giulivi, C., Poderoso, J. J., and Boveris, A. (1998) J. Biol. Chem. 273, 11038-11043). Here we report on the possible involvement of mtNOS in apoptosis. We show that uptake of Ca(2+) by mitochondria triggers mtNOS activity and causes the release of cytochrome c from isolated mitochondria in a Bcl-2-sensitive manner. mtNOS-induced cytochrome c release was paralleled by increased lipid peroxidation. The release of cytochrome c as well as increase in lipid peroxidation were prevented by NOS inhibitors, a superoxide dismutase mimic, and a peroxynitrite scavenger. We show that mtNOS-induced cytochrome c release is not mediated via the mitochondrial permeability transition pore because the release was aggravated by cyclosporin A and abolished by blockade of mitochondrial calcium uptake by ruthenium red. We conclude that, upon Ca(2+)-induced mtNOS activation, peroxynitrite is formed within mitochondria, which causes the release of cytochrome c from isolated mitochondria, and we propose a mechanism by which elevated Ca(2+) levels induce apoptosis.

In the present study we show thatN-acetylsphingosine (C2-ceramide),N-hexanoylsphingosine (C6-ceramide), and, to a much lesser extent, C2-dihydroceramide induce cytochromec (cyto c) release from isolated rat liver mitochondria. Ceramide-induced cyto c release is prevented by preincubation of mitochondria with a low concentration (40 nm) of Bcl-2. The release takes place when cytoc is oxidized but not when it is reduced. Upon cytoc loss, mitochondrial oxygen consumption, mitochondrial transmembrane potential (ΔΨ), and Ca2+ retention are diminished. Incubation with Bcl-2 prevents, and addition of cytoc reverses the alteration of these mitochondrial functions. In ATP-energized mitochondria, ceramides do not alter ΔΨ, neither when cyto c is oxidized nor when it is reduced, ruling out a nonspecific disturbance by ceramides of mitochondrial membrane integrity. Furthermore, ceramides decrease the reducibility of cytoc. We conclude that the apoptogenic properties of ceramides are in part mediated via their interaction with mitochondrial cytoc followed by its release and that the redox state of cytoc influences its detachment by ceramide from the inner mitochondrial membrane.

International audience; BACKGROUND INFORMATION: ATP is the main transmitter stored and released from astrocytes under physiological and pathological conditions. Morphological and functional evidence suggest that besides secretory granules, secretory lysosomes release ATP. However, the molecular mechanisms involved in astrocytic lysosome fusion remain still unknown. RESULTS: In the present study, we identify tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP, also called VAMP7) as the vesicular SNARE which mediates secretory lysosome exocytosis, contributing to release of both ATP and cathepsin B from glial cells. We also demonstrate that fusion of secretory lysosomes is triggered by slow and locally restricted calcium elevations, distinct from calcium spikes which induce the fusion of glutamate-containing clear vesicles. Downregulation of TI-VAMP/VAMP7 expression inhibited the fusion of ATP-storing vesicles and ATP-mediated calcium wave propagation. TI-VAMP/VAMP7 downregulation also significantly reduced secretion of cathepsin B from glioma. CONCLUSIONS: Given that sustained ATP release from glia upon injury greatly contributes to secondary brain damage and cathepsin B plays a critical role in glioma dissemination, TI-VAMP silencing can represent a novel strategy to control lysosome fusion in pathological conditions.

Extracellular nucleotides are pleiotropic regulators of mammalian cell function. Adenosine triphosphate (ATP) released from CD4 + helper T cells upon stimulation of the T cell receptor (TCR) contributes in an autocrine manner to the activation of mitogen-activated protein kinase (MAPK) signaling through purinergic P2X receptors. Increased expression of p2rx7 , which encodes the purinergic receptor P2X7, is part of the transcriptional signature of immunosuppressive CD4 + CD25 + regulatory T cells (T regs ). Here, we show that the activation of P2X7 by ATP inhibits the suppressive potential and stability of T regs . The inflammatory cytokine interleukin-6 (IL-6) increased ATP synthesis and P2X7-mediated signaling in T regs , which induced their conversion to IL-17–secreting T helper 17 (T H 17) effector cells in vivo. Moreover, pharmacological antagonism of P2X receptors promoted the cell-autonomous conversion of naive CD4 + T cells into T regs after TCR stimulation. Thus, ATP acts as an autocrine factor that integrates stimuli from the microenvironment and cellular energetics to tune the developmental and immunosuppressive program of the T cell in adaptive immune responses.

in thrombotic events with heparin-coated Palmaz-Schatz stents in normal porcine coronary arteries. Circulation 1996;93:423-30. 41. Burchenal JEB, Marks DS, Mann JT, Schweiger MJ, Rothman MT, Ganz P, et al. Effect of direct thrombin inhibition with Bivalirudin (Hirulog) on restenosis after coronary angioplasty. Am J Cardiol 1998;82:511-5. 42. van de WerfF. More evidence for a beneficial effect ofplatelet glycoprotein IIb/IIIa-blockade during coronary interventions: latest results from the EPILOG and CAPTURE trials. Circulation 1996;93:637. 43. Aggarwal RK, Ireland DC, Azrin MA, Ezekowitz MD, deBono DP, Gershlick Ah. Antithrombotic potential of polymer-coated stents eluting platelet glycoprotein I Ib / I I Ia receptor antibody. Circulation 1996;94:3311-7. 44. Sollott SJ, Cheng L, Pauly RR, Jenkins GM, Monticone RE, Kuzuya M, et aft. Taxol inhibits neointimal smooth muscle cell accumulation after angioplasty in the rat. J Clin Invest 1995;95:1869-76. 45. Axel DI, Kunert W, Goggelmann C, OberhoffM, Herdeg C, Kuttner A, et al. Paclitaxel inhibits arterial smooth muscle cell proliferation and migration in vitro and in vivo using local drug delivery. Circulation 1997;96:636-45.

The regulation of filopodia plays a crucial role during neuronal development and synaptogenesis. Axonal filopodia, which are known to originate presynaptic specializations, are regulated in response to neurotrophic factors. The structural components of filopodia are actin filaments, whose dynamics and organization are controlled by ensembles of actin-binding proteins. How neurotrophic factors regulate these latter proteins remains, however, poorly defined. Here, using a combination of mouse genetic, biochemical, and cell biological assays, we show that genetic removal of Eps8, an actin-binding and regulatory protein enriched in the growth cones and developing processes of neurons, significantly augments the number and density of vasodilator-stimulated phosphoprotein (VASP)-dependent axonal filopodia. The reintroduction of Eps8 wild type (WT), but not an Eps8 capping-defective mutant, into primary hippocampal neurons restored axonal filopodia to WT levels. We further show that the actin barbed-end capping activity of Eps8 is inhibited by brain-derived neurotrophic factor (BDNF) treatment through MAPK-dependent phosphorylation of Eps8 residues S624 and T628. Additionally, an Eps8 mutant, impaired in the MAPK target sites (S624A/T628A), displays increased association to actin-rich structures, is resistant to BDNF-mediated release from microfilaments, and inhibits BDNF-induced filopodia. The opposite is observed for a phosphomimetic Eps8 (S624E/T628E) mutant. Thus, collectively, our data identify Eps8 as a critical capping protein in the regulation of axonal filopodia and delineate a molecular pathway by which BDNF, through MAPK-dependent phosphorylation of Eps8, stimulates axonal filopodia formation, a process with crucial impacts on neuronal development and synapse formation.

T cell receptor (TCR) stimulation results in the influx of Ca(2+), which is buffered by mitochondria and promotes adenosine triphosphate (ATP) synthesis. We found that ATP released from activated T cells through pannexin-1 hemichannels activated purinergic P2X receptors (P2XRs) to sustain mitogen-activated protein kinase (MAPK) signaling. P2XR antagonists, such as oxidized ATP (oATP), blunted MAPK activation in stimulated T cells, but did not affect the nuclear translocation of the transcription factor nuclear factor of activated T cells, thus promoting T cell anergy. In vivo administration of oATP blocked the onset of diabetes mediated by anti-islet TCR transgenic T cells and impaired the development of colitogenic T cells in inflammatory bowel disease. Thus, pharmacological inhibition of ATP release and signaling could be beneficial in treating T cell-mediated inflammatory diseases.

Regulation of brain-derived neurotrophic factor (BDNF) secretion plays a critical role in long-term potentiation (LTP). It is generally thought that the supply for this secretion is newly synthesized BDNF targeted to the synapse. Here we provide evidence that hippocampal neurons additionally recycle BDNF for activity-dependent secretion. Exogenously applied BDNF is internalized by cultured neurons and rapidly becomes available for activity-dependent secretion, which is controlled by the same mechanisms that regulate the secretion of newly synthesized BDNF. Moreover, BDNF recycling replaced the new synthesis pathway in mediating the maintenance of LTP in hippocampal slices: the late phase LTP, which is abolished by protein synthesis inhibition, was rescued in slices preincubated with BDNF. Thus, endocytosed BDNF is fed back to the activity-dependent releasable pool required for LTP maintenance.

ATP has been indicated as a primary factor in microglial response to brain injury and inflammation. By acting on different purinergic receptors 2, ATP is known to induce chemotaxis and stimulate the release of several cytokines from these cells. The activation of purinergic receptors 2 in microglia can be triggered either by ATP deriving from dying cells, at sites of brain injury or by ATP released from astrocytes, in the absence of cell damage. By the use of a biochemical approach integrated with video microscopy experiments, we investigated the functional consequences triggered in microglia by ATP released from mechanically stimulated astrocytes, in mixed glial cocultures. Astrocyte-derived ATP induced in nearby microglia the formation and the shedding of membrane vesicles. Vesicle formation was inhibited by the ATP-degrading enzyme apyrase or by P2X7R antagonists. Isolation of shed vesicles, followed by IL-1β evaluation by a specific ELISA revealed the presence of the cytokine inside the vesicular organelles and its subsequent efflux into the extracellular medium. IL-1β efflux from shed vesicles was enhanced by ATP stimulation and inhibited by pretreatment with the P2X7 antagonist oxidized ATP, thus indicating a crucial involvement of the pore-forming P2X7R in the release of the cytokine. Our data identify astrocyte-derived ATP as the endogenous factor responsible for microvesicle shedding in microglia and reveal the mechanisms by which astrocyte-derived ATP triggers IL-1β release from these cells.

AMPA-type glutamate receptors play a key role in mediating postsynaptic responses of excitatory neurotransmitters. It is now well accepted that AMPA receptors are also present at the presynapse, where they are thought to modulate neurotransmitter release. However, the mechanisms through which they control synaptic vesicle traffic have remained elusive. We used cultured hippocampal neurons and growth cone particles prepared from fetal rat brain to investigate the functional role of presynaptic AMPA receptors. We show here that stimulation of presynaptic AMPA receptors induces activation of mitogen-activated protein kinase (MAPK) through a nonreceptor tyrosine kinase-dependent and Na+/Ca2+-independent mechanism. This pathway is activated predominantly in axonal growth cones compared with the somatodendritic compartment. After stimulation of presynaptic AMPA receptors, synapsin I is phosphorylated at MAPK-specific sites. These events are paralleled by a prominent increase in evoked synaptic vesicle recycling that is blocked by the specific MAPK inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one. Similarly, in synaptosomes isolated from adult brain, AMPA stimulation induces MAPK activation and phosphorylation of synapsin I at MAPK-dependent sites and enhances significantly synaptic vesicle recycling. These results reveal a novel pathway for activation of presynaptic MAPK and suggest a role of this pathway in the regulation of short-term presynaptic plasticity.

Although vascular endothelial growth factor (VEGF) has been described as a potent angiogenic stimulus, its application in therapy remains difficult: blood vessels formed by exposure to VEGF tend to be malformed and leaky. In nature, the principal form of VEGF possesses a binding site for ECM components that maintain it in the immobilized state until released by local cellular enzymatic activity. In this study, we present an engineered variant form of VEGF, α 2 PI 1–8 -VEGF 121 , that mimics this concept of matrix-binding and cell-mediated release by local cell-associated enzymatic activity, working in the surgically-relevant biological matrix fibrin. We show that matrix-conjugated α 2 PI 1–8 -VEGF 121 is protected from clearance, contrary to native VEGF 121 mixed into fibrin, which was completely released as a passive diffusive burst. Grafting studies on the embryonic chicken chorioallantoic membrane (CAM) and in adult mice were performed to assess and compare the quantity and quality of neovasculature induced in response to fibrin implants formulated with matrix-bound α 2 PI 1–8 -VEGF 121 or native diffusible VEGF 121 . Our CAM measurements demonstrated that cell-demanded release of α 2 PI 1–8 -VEGF 121 increases the formation of new arterial and venous branches, whereas exposure to passively released wild-type VEGF 121 primarily induced chaotic changes within the capillary plexus. Specifically, our analyses at several levels, from endothelial cell morphology and endothelial interactions with periendothelial cells, to vessel branching and network organization, revealed that α 2 PI 1–8 -VEGF 121 induces vessel formation more potently than native VEGF 121 and that those vessels possess more normal morphologies at the light microscopic and ultrastructural level. Permeability studies in mice validated that vessels induced by α 2 PI 1–8 -VEGF 121 do not leak. In conclusion, cell-demanded release of engineered VEGF 121 from fibrin implants may present a therapeutically safe and practical modality to induce local angiogenesis.

Vascular endothelial growth factor (VEGF) is a key factor in endothelial cell biology and blood vessel formation and a candidate therapeutic for the stimulation of angiogenesis-dependent tissue regeneration. The objective of this study was to confer the angiogenic activity of VEGF(121) upon the biomaterial fibrin, a natural substrate for endothelial cell growth and clinically accepted as 'fibrin glue'. To achieve this, we engineered fibrin-based hydrogels that were covalently modified with VEGF(121). Our laboratory has recently developed novel methodology that allows the covalent incorporation of exogenous bioactive peptides by the transglutaminase activity of factor XIIIa into fibrin during coagulation. Here, this ability of factor XIIIa to crosslink additional proteins within fibrin was employed to covalently incorporate VEGF(121). By recombinant DNA methodology, a mutant VEGF(121) variant, alpha(2)-PI(1--8)-VEGF(121), which contains an additional factor XIIIa substrate sequence NQEQVSPL at the aminoterminus, was expressed in E. coli. In soluble form, the mutant protein fully retained its mitogenic activity for endothelial cells. Using (125)I-labeled alpha(2)-PI(1--8)-VEGF(121), its covalent incorporation and the efficiency of incorporation into fibrin was demonstrated and characterized. The immobilized, fibrin-conjugated VEGF(121) protein remained an active and very efficient mitogen for human endothelial cells grown on two-dimensional VEGF(121)-modified fibrin surfaces, and the incorporation of increasing amounts of alpha(2)-PI(1--8)-VEGF(121) resulted in dose-dependent enhancement of endothelial cell growth. The VEGF-modified fibrin matrices can be formed as injectable gels in a single-step reaction under physiological conditions in vivo. When used as a ingrowth matrix, such VEGF incorporating materials could be useful in a variety of clinical situations that require an angiogenic response into an ischemic region or inplant.

Wiskott-Aldrich Syndrome (WAS) is an X-linked immunodeficiency caused by mutations of the gene encoding for WASp, a protein involved in cytoskeletal reorganization of hematopoietic cells. WAS is characterized by microthrombocytopenia, eczema, infections, tumors and high rate of autoimmunity. WASp negative T cells have been extensively studied, while the role of WASp in B cells has been less characterized. Analysis of serum from adult was-/- mice showed increased level of Igs as compared to wt mice, in particular in IgG2a subclass. This result was further confirmed by ELISpot assays performed on sorted Marginal Zone (MZ) and Follicular (FO) B cells from wt and was-/- mice. The presence of autoreactive B cells was supported by the high titers of autoantibodies against dsDNA and tissue Ags in was-/- mice. In in vitro proliferation assays, was-/- B cells are hyper-proliferating to BCR-dependent (IgM) and Toll Like Receptors (TLRs)-dependent stimuli (LPS, CpG) in comparison to wt B cells, although no difference in the expression of TLRs was found. Preliminary results on Ca2+ flux upon CpG stimulus revealed increased Ca2+ mobilization in was-/- MZ B cell as compared to wt MZ B cells. Our data suggest that WASp absence affects B cell function and further studies are ongoing to better characterize BCR and TLR signaling pathways and intracellular trafficking in order to understand the contribution of B cells in autoimmune manifestations found in WAS patients.

Recently, a new system of astrocyte-neurone glutamatergic signalling has been identified. It is started in astrocytes by ectocellular, CD38-catalysed conversion of NAD(+) to the calcium mobilizer cyclic ADP-ribose (cADPR). This is then pumped by CD38 itself into the cytosol where the resulting free intracellular Ca(2+) concentration [Ca(2+)](i) transients elicit an increased release of glutamate, which can induce an enhanced Ca(2+) response in neighbouring neurones. Here, we demonstrate that co-culture of either cortical or hippocampal astrocytes with neurones results in a significant overexpression of astrocyte CD38 both on the plasma membrane and intracellularly. The causal role of neurone-released glutamate in inducing overexpression of astrocyte CD38 is demonstrated by two observations: first, in the absence of neurones, induction of CD38 in pure astrocyte cultures can be obtained with glutamate and second, it can be prevented in co-cultures by glutamate receptor antagonists. The neuronal glutamate-mediated effect of neurones on astrocyte CD38 expression is paralleled by increased intracellular cADPR and [Ca(2+)](i) levels, both findings indicating functionality of overexpressed CD38. These results reveal a new neurone-to-astrocyte glutamatergic signalling based on the CD38/cADPR system, which affects the [Ca(2+)](i) in both cell types, adding further complexity to the bi-directional patterns of communication between astrocytes and neurones.

Outgrowth of the dendrites and the axon is the basis of the establishment of the neuronal shape, and it requires addition of new membrane to both growing processes. It is not yet clear whether one or two exocytotic pathways are responsible for the respective outgrowth of axons and dendrites. We have previously shown that tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) defines a novel network of tubulovesicular structures present both at the leading edge of elongating dendrites and axons of immature hippocampal neurons developing in primary culture and that TI-VAMP is an essential protein for neurite outgrowth in PC12 cells. Here we show that the expression of the N-terminal domain of TI-VAMP inhibits the outgrowth of both dendrites and axons in neurons in primary culture. This effect is more prominent at the earliest stages of the development of neuronsin vitro. Expression of the N-terminal domain deleted form of TI-VAMP has the opposite effect. This constitutively active form of TI-VAMP localizes as the endogenous protein, particularly concentrating at the leading edge of growing axons. Our results suggest that a common exocytotic mechanism that relies on TI-VAMP mediates both axonal and dendritic outgrowth in developing neurons.