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Mesoporous bioactive glasses (MBGs) are bioceramics designed to induce bone tissue regeneration and very useful materials with the ability to act as drug delivery systems. MBGs can be implanted in contact with bone tissue in different ways, as particulate material, in 3D scaffolds or as nanospheres. In this work, we assessed the effects of particles of mesoporous bioactive glass MBG-75S and mesoporous nanospheres NanoMBG-75S on RAW 264.7 and J774A.1 macrophages, which present different sensitivity and are considered as ideal models for the study of innate immune response. After evaluating several cellular parameters (morphology, size, complexity, proliferation, cell cycle and intracellular content of reactive oxygen species), the action of MBG-75S particles and NanoMBG-75S on the polarization of these macrophages towards the proinflammatory (M1) or reparative (M2) phenotype was determined by the expression of specific M1 (CD80) and M2 (CD206, CD163) markers. We previously measured the adsorption of albumin and fibrinogen on MBG-75S particles and the production of proinflammatory cytokines as TNF-{\alpha} and IL-6 by macrophages in response to these particles. This comparative study demonstrates that particles of mesoporous bioactive glass MBG-75S and mesoporous nanospheres NanoMBG-75S allow the appropriated development and function of RAW 264.7 and J774A.1 macrophages and do not induce polarization towards the M1 pro-inflammatory phenotype. Therefore, considering that these mesoporous biomaterials offer the possibility of loading drugs into their pores, the results obtained indicate their high potential for use as drug-delivery systems in bone repair and osteoporosis treatments without triggering an adverse inflammatory response., Comment: 29 pages, 7 figures

Graphene and its derivatives are very promising nanomaterials for biomedical applications and are proving to be very useful for the preparation of scaffolds for tissue repair. The response of immune cells to these graphene-based materials (GBM) appears to be critical in promoting regeneration, thus, the study of this response is essential before they are used to prepare any type of scaffold. Another relevant factor is the variability of the GBM surface chemistry, namely the type and quantity of oxygen functional groups, which may have an important effect on cell behavior. The response of RAW-264.7 macrophages to graphene oxide (GO) and two types of reduced GO, rGO15 and rGO30, obtained after vacuum-assisted thermal treatment of 15 and 30 min, respectively, was evaluated by analyzing the uptake of these nanostructures, the intracellular content of reactive oxygen species, and specific markers of the proinflammatory M1 phenotype, such as CD80 expression and secretion of inflammatory cytokines TNF-α and IL-6. Our results demonstrate that GO reduction resulted in a decrease of both oxidative stress and proinflammatory cytokine secretion, significantly improving its biocompatibility and potential for the preparation of 3D scaffolds able of triggering the appropriate immune response for tissue regeneration.

10 p.-6 fig.-2 tab., Macrophages play a key role in nanoparticle removal and are primarily responsible for their uptake and trafficking in vivo. Due to their functional plasticity, macrophages display a spectrum of phenotypes between two extremes indentified as pro-inflammatory M1 and reparative M2 macrophages, characterized by the expression of specific cell surface markers and the secretion of different cytokines. The influence of graphene oxide (GO) nanosheets functionalized with poly(ethylene glycol-amine) and labelled with fluorescein isothiocyanate (FITC-PEG-GO) on polarization of murine peritoneal macrophages towards M1 and M2 phenotypes was evaluated in basal and stimulated conditions by flow cytometry and confocal microscopy through the expression of different cell markers: CD80 and iNOS as M1 markers, and CD206 and CD163 as M2 markers. Although FITC-PEG-GO did not induce M1 or M2 macrophage polarization after 24 and 48 h in basal conditions, this nanomaterial decreased the percentage of M2 reparative macrophages. We have also compared control macrophages with macrophages that have or have not taken up FITC-PEG-GO after treatment with these nanosheets (GO+ and GO− cells, respectively). The CD80 expression diminished in GO+ macrophages after 48 h of GO treatment but the CD206 expression in GO+ population showed higher values than in both GO- population and control macrophages. In the presence of pro-inflammatory stimuli (LPS and IFN-γ), a significant decrease of CD80+ cells was observed after treatment with GO. This nanomaterial also induced significant decreases of CD206+ and CD163+ cells in the presence of reparative stimulus (IL-4). The CD80, iNOS and CD206 expression was lower in both GO− and GO+ cells than in control macrophages. However, higher CD163 expression was obtained in both GO− and GO+ cells in comparison with control macrophages. All these facts suggest that FITC-PEG-GO uptake did not induce the macrophage polarization towards the M1 pro-inflammatory phenotype, promoting the control of the M1/M2 balance with a slight shift towards M2 reparative phenotype involved in tissue repair, ensuring an appropriate immune response to these nanosheets., This study was supported by research grant from the Spanish Ministerio de Economía y Competitividad (MAT2016-75611-R AEI/FEDER, UE). M.C. acknowledges the financial support from the FCT [SFRH/BPD/101468/2014 Post-Doctoral grant]. J.M. Rojo is supported by Grant PI13/01809 from “AES, Plan Estatal I+D+i, ISCIII-Subdirección General de Evaluación y Fomento de la Investigación, Ministerio de Economía y Competitividad (MINECO).

Angiogenic biomaterials for bone repair are being designed to promote vascularization and optimize tissue regeneration. The use of nanoparticles of bioactive materials loaded with different drugs represents an interesting strategy to stimulate osteogenesis and angiogenesis and to inhibit bone resorption. Ipriflavone (IP) prevents osteoporosis by inhibiting osteoclast activity and promoting preosteoblast differentiation into mature osteoblasts. Since endothelial progenitor cells (EPCs) are involved in the formation of blood vessels which are necessary for tissue regeneration, the isolation and characterization of porcine EPCs have been carried out in this work to evaluate the in vitro effects of unloaded (NanoMBGs) and IP-loaded nanospheres (NanoMBG-IPs) designed to stimulate osteogenesis. Because different signals between vascular and nonvascular cells are also essential to initiate angiogenic events, the potential modulating role of macrophages has been also evaluated by studying the expression of vascular endothelial growth factor receptor 2 (VEFGR2) as a specific marker for EPC differentiation under different culture conditions: a) EPCs in monoculture treated with NanoMBGs or NanoMBG-IPs, b) EPCs treated with conditioned media from basal, proinflammatory M1 and reparative M2 macrophages previously treated with NanoMBGs or NanoMBG-IPs, c) EPCs cocultured with macrophages in the presence of NanoMBGs or NanoMBG-IPs, and d) EPCs cocultured with M2d angiogenic macrophages. Moreover, the endocytic mechanisms by which these nanospheres are incorporated by EPCs have been identified by using six endocytosis inhibitors (i.e. wortmannin, genistein, cytochalasin B, cytochalasin D, phenylarsine oxide and chlorpromazine) and before the addition of NanoMBGs labeled with fluorescein isothiocyanate. The results evidence the great potential of both NanoMBGs and NanoMBG-IPs to enhance VEFGR2 expression, directly related to angiogenesis, after intracellular incorporation by EPCs through different endocytic mechanisms including clathrin-dependent endocytosis, as the main entry mechanism, but also phagocytosis and caveolae-mediated uptake. The treatment of EPCs with culture media from basal, M1 and M2 macrophages and the development of cocultures of EPCs with macrophages in the absence and presence of these nanomaterials have also confirmed the maintenance of their angiogenic effect on EPCs even in the presence of phagocytic cells.

35 p.-6 fig.-2 tab., The use of nanoparticles for intracellular drug delivery could reduce the toxicity and side effects of the drug but, the uptake of these nanocarriers could induce adverse effects on cells and tissues after their incorporation. Macrophages play a central role in host defense and are responsible for in vivo nanoparticle trafficking. Assessment of their defense capacity against pathogenic micro-organisms after nanoparticle uptake, is necessary to prevent infections associated with nanoparticle therapies. In this study, the effects of hollow mesoporous SiO2-CaO nanospheres labeled with fluorescein isothiocyanate (FITC-NanoMBGs) on the function of peritoneal macrophages was assessed by measuring their ability to phagocytize Candida albicans expressing a red fluorescent protein. Two macrophage/fungus ratios (MOI 1 and MOI 5) were used and two experimental strategies were carried out: a) pretreatment of macrophages with FITC-NanoMBGs and subsequent fungal infection; b) competition assays after simultaneous addition of fungus and nanospheres. Macrophage pro-inflammatory phenotype markers (CD80 expression and interleukin 6 secretion) were also evaluated. Significant decreases of CD80+ macrophage percentage and interleukin 6 secretion were observed after 30 min, indicating that the simultaneous incorporation of NanoMBG and fungus favors the macrophage non-inflammatory phenotype. The present study evidences that the uptake of these nanospheres in all the studied conditions does not alter the macrophage function. Moreover, intracellular FITC-NanoMBGs induce a transitory increase of the fungal phagocytosis by macrophages at MOI 1 and after a short time of interaction. In the competition assays, as the intracellular fungus quantity increased, the intracellular FITC-NanoMBG content decreased in a MOI- and time-dependent manner. These results have confirmed that macrophages clearly distinguish between inert material and the live yeast in a dynamic intracellular incorporation. Furthermore, macrophage phagocytosis is a critical determinant to know their functional state and a valuable parameter to study the nanomaterial / macrophages / Candida albicans interface., This study was supported by research grants from the Ministerio de Economía y Competitividad, Agencia Estatal de Investigación (AEI), Fondo Europeo de Desarrollo Regional (FEDER) (MAT2016-75611-R AEI/FEDER, UE) and 4129533-FEI-EU-2018 Project. MVR acknowledges funding from the European Research Council (Advanced Grant VERDI; ERC-2015-AdG Proposal No. 694160).

The decellularized extracellular matrix (ECM) obtained from human and porcine adipose tissue (AT) is currently used to prepare regenerative medicine bio-scaffolds. However, the influence of these natural biomaterials on host immune response is not yet deeply understood. Since macrophages play a key role in the inflammation/healing processes due to their high functional plasticity between M1 and M2 phenotypes, the evaluation of their response to decellularized ECM is mandatory. It is also necessary to analyze the immunocompetence of macrophages after contact with decellularized ECM materials to assess their functional role in a possible infection scenario. In this work, we studied the effect of four decellularized adipose matrices (DAMs) obtained from human and porcine AT by enzymatic or chemical methods on macrophage phenotypes and fungal phagocytosis. First, a thorough biochemical characterization of these biomaterials by quantification of remnant DNA, lipids, and proteins was performed, thus indicating the efficiency and reliability of both methods. The proteomic analysis evidenced that some proteins are differentially preserved depending on both the AT origin and the decellularization method employed. After exposure to the four DAMs, specific markers of M1 proinflammatory and M2 anti-inflammatory macrophages were analyzed. Porcine DAMs favor the M2 phenotype, independently of the decellularization method employed. Finally, a sensitive fungal phagocytosis assay allowed us to relate the macrophage phagocytosis capability with specific proteins differentially preserved in certain DAMs. The results obtained in this study highlight the close relationship between the ECM biochemical composition and the macrophage’s functional role. This work has been supported by the European Union’s Horizon 2020 Research and Innovation Programme (H2020-FETOPEN-2018-2020, NeuroStimSpinal Project, Grant Agreement No. 829060). M.C. acknowledges the European Union0s Horizon 2020 Research and Innovation Programme for her contract under the NeuroStimSpinal Project. LC is grateful to the Universidad Complutense de Madrid for an UCM fellowship.

Graphene oxide (GO) is a new nanomaterial with different potential biomedical applications due to its excellent physicochemical properties and ease of surface functionalization. Macrophages play key roles in the control of fungal infections preventing invasive candidiasis by both limiting the growth of the opportunistic fungal pathogen Candida albicans and activating other immune effector cells. In order to know if macrophages maintain their immunocompetence against this microorganism after GO uptake, we have evaluated the interactions at the interface of GO nanosheets, macrophages and Candida albicans. Poly (ethylene glycol-amine)-derivatized GO nanosheets labelled with fluorescein isothiocyanate (FITC-PEG-GO), were efficiently taken up by peritoneal macrophages inducing a significant increase of C. albicans phagocytosis by both pro-inflammatory macrophages (M1/stimulated with LPS/IFN-γ) and reparative macrophages (M2/stimulated with IL-4). On the other hand, after FITC-PEG-GO treatment and C. albicans infection, the percentages of GO+ macrophages diminished when Candida uptake increased in every condition (macrophages with no stimuli, M1 and M2 macrophages), thus suggesting the exocytosis of this nanomaterial as a dynamic mechanism favoring fungal phagocytosis. For the first time, we have analyzed the effects of PEG-GO nanosheets on Candida albicans killing by unstimulated, M1 and M2 macrophages, evidencing that intracellular GO modulates the macrophage candidacidal activity in a multiplicity of infection (MOI) dependent manner. At MOI 1, the high intracellular GO levels increase the fungicidal activity of basal and stimulated macrophages. At MOI 5, as intracellular GO decreases, the previous pro-inflammatory or reparative stimulus predefines the killing ability of macrophages. In summary, GO treatment enhances classical M1 macrophage activation, important for pathogen eradication, and diminishes alternative activation of M2 macrophages, thus decreasing fungal persistence and avoiding chronic infectious diseases.

Graphene-based materials are revealing a great promise for biomedical applications and demonstrating attractiveness for neural repair. In the context of neural tissue damage, the dialogue between neural and immune cells appears critical for driving regeneration, thus making the understanding of their relations pivotal. Herein, the acute response of RAW-264.7 macrophages on nanostructured reduced graphene oxide (rGO) microfibers has been evaluated through the analysis of cell parameters including proliferation, viability, intracellular content of reactive oxygen species, cell cycle, apoptosis, and cell size and complexity. The influence of the direct contact of rGO microfibers on their polarization towards M1 and M2 phenotypes has been studied by analyses of both M1 (CD80) and M2 (CD163) markers and the secretion of the inflammatory cytokines TNF-α and IL-6. Finally, the capability of these rGO microfibers to regulate neural stem cell differentiation has been also evaluated. Findings reveal that rGO microfibers inhibit the proliferation of RAW-264.7 macrophages without affecting their viability and cell cycle profiles. The presence of M1 and M2 macrophages on these microfibers was confirmed after 24 and 48 h, respectively, accompanied by a decrease in TNF-α and an increase in IL-6 cytokine secretion. These rGO microfibers were also able to support the formation of a highly interconnected neural culture composed of both neurons (map2+ cells) and glial cells (vimentin+ cells). These findings encourage further investigation of these microfibers as attractive biomaterials to interact with immune and neural cells, attempting to support wound healing and tissue repair after implantation.

Due to their specific mesoporous structure and large surface area, mesoporous bioactive glasses (MBGs) possess both drug-delivery ability and effective ionic release to promote bone regeneration by stimulating osteogenesis and angiogenesis. Macrophages secrete mediators that can affect both processes, depending on their phenotype. In this work, the action of ion release from MBG-75S, with a molar composition of 75SiO2-20CaO-5P2O5, on osteogenesis and angiogenesis and the modulatory role of macrophages have been assessed in vitro with MC3T3-E1 pre-osteoblasts and endothelial progenitor cells (EPCs) in monoculture and in coculture with RAW 264.7 macrophages. Ca2+, phosphorous, and silicon ions released from MBG-75S were measured in the culture medium during both differentiation processes. Alkaline phosphatase activity and matrix mineralization were quantified as the key markers of osteogenic differentiation in MC3T3-E1 cells. The expression of CD31, CD34, VEGFR2, eNOS, and vWF was evaluated to characterize the EPC differentiation into mature endothelial cells. Other cellular parameters analyzed included the cell size and complexity, intracellular calcium, and intracellular content of the reactive oxygen species. The results obtained indicate that the ions released by MBG-75S promote osteogenesis and angiogenesis in vitro, evidencing a macrophage inhibitory role in these processes and demonstrating the high potential of MBG-75S for the preparation of implants for bone regeneration.

The differences in the response of human Saos-2 osteoblasts to nanocomposites of poly(vinyl alcohol) (PVA) and 1.5 wt.% graphene oxide (GO) prepared by covalent linking (PVA/GO-c) and simple blending (PVA/GO-m) have been evaluated through different biocompatibility parameters. The effects produced on osteoblasts by these two nanocomposites were analysed in parallel and compared with the direct action of GO and with the effect of PVA films without GO. The intracellular content of reactive oxygen species (ROS) and the levels of interleukin-6 (IL-6) were measured to evaluate oxidative stress induction and protective response, respectively. The results demonstrate that the combination of GO with PVA reduces both the proliferation delay and the internal cell complexity alterations induced by GO on human osteoblasts. Moreover, the covalent attachment of GO to the PVA chains increases both cell viability and IL-6 levels, reducing both apoptosis and intracellular ROS content when compared to simple blending of both materials. The use of this strategy to modulate the biointerface reduces the toxic effects of graphene while preserving the reinforcement characteristics for application in tissue engineering scaffolds, and has enormous interest for polymer/graphene biomaterials development.

Silicon substituted and nanocrystalline hydroxyapatites have attracted the attention of many researchers due to their up-regulation in osteoblast cell metabolism and enhanced bioreactivity, respectively. On the other hand, the biomaterial success or failure depends ultimately on the immune response triggered after its implantation. Macrophages are the main components of the innate immune system with an important role in healing and tissue remodelling due to their remarkable functional plasticity, existing in a whole spectrum of functional populations with varying phenotypic features. The effects of nanocrystalline hydroxyapatite (nano-HA) and nanocrystalline silicon substituted hydroxyapatite (nano-SiHA) on the macrophage populations defined as pro-inflammatory (M1) and reparative (M2) phenotypes have been evaluated in the present study using RAW 264.7 cells and mouse peritoneal macrophages as in vitro models. M1 and M2 macrophage phenotypes were characterized by flow cytometry and confocal microscopy by the expression of CD80 and CD163, known as M1 and M2 markers, respectively. The polarization of primary macrophages towards the M1 or M2 phenotype was induced with the pro-inflammatory stimulus LPS or the anti-inflammatory stimulus IL-10, respectively, evaluating the biomaterial effects under these conditions. Our results show that both nano-HA and nano-SiHA favour the macrophage polarization towards an M2 reparative phenotype, decreasing M1 population and ensuring an appropriate response in the implantation site of these biomaterials designed for bone repair and bone tissue engineering.

The preparation of graphene-based nanomaterials (GBNs) with appropriate stability and biocompatibility is crucial for their use in biomedical applications. In this work, three GBNs differing in size and/or functionalization have been synthetized and characterized, and their in vitro biological effects were compared. Pegylated graphene oxide (GO-PEG, 200–500 nm) and flavin mononucleotide-stabilized pristine graphene with two different sizes (PG-FMN, 200–400 nm and 100–200 nm) were administered to macrophages, chosen as cellular model due to their key role in the processing of foreign materials and the regulation of inflammatory responses. The results showed that cellular uptake of GBNs was mainly influenced by their lateral size, while the inflammatory potential depended also on the type of functionalization. PG-FMN nanomaterials (both sizes) triggered significantly higher nitric oxide (NO) release, together with some intracellular metabolic changes, similar to those induced by the prototypical inflammatory stimulus LPS. NMR metabolomics revealed that macrophages incubated with smaller PG-FMN displayed increased levels of succinate, itaconate, phosphocholine and phosphocreatine, together with decreased creatine content. The latter two variations were also detected in cells incubated with larger PG-FMN nanosheets. On the other hand, GO-PEG induced a decrease in the inflammatory metabolite succinate and a few other changes distinct from those seen in LPS-stimulated macrophages. Assessment of TNF-α secretion and macrophage surface markers (CD80 and CD206) further corroborated the low inflammatory potential of GO-PEG. Overall, these findings revealed distinct phenotypic and metabolic responses of macrophages to different GBNs, which inform on their immunomodulatory activity and may contribute to guide their therapeutic applications., This work was developed in the scope of the projects CICECO-Aveiro Institute of Materials, FCT Ref. UID/CTM/50011/2019, and CESAM, FCT Ref. UID/AMB/50017/2019, financed by national funds through FCT/MCTES, Portugal. The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project Nº 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). Further acknowledgments are due to Bruker BioSpinGmbH and the European Union Framework Programme for Research and Innovation HORIZON 2020, under the TEAMING Grant agreement No 739572 - The Discoveries CTR. I.F.D. and H.O. acknowledge FCT/MCTES for research contracts under the Programs ‘Investigador FCT’ and Stimulus to Scientific Employment (CEECIND/04050/2017), respectively, and M.C. acknowledges the FCT-awarded research grant SFRH/BPD/101468/2014. This research was also supported by the Spanish Ministerio de Economía y Competitividad (MAT2016-75611-R AEI/FEDER, UE). M.A.-V., S.V.-R. and J.I.P. acknowledge financial support from the Spanish Ministerio de Economía y Competitividad (MINECO) and the European Regional Development Fund (ERDF) through project MAT2015-69844-R, and from the Spanish Ministerio de Ciencia, Innovación y Universidades, the Spanish Agencia Estatal de Investigación and ERDF, through project RTI2018-100832-B-I00. Partial funding by Plan de Ciencia, Tecnología e Innovación (PCTI) 2013-2017 del Principado de Asturias and ERDF (project IDI/2018/000233) is also acknowledged.

The osteogenic and angiogenic responses to metal macroporous scaffolds coated with silicon substituted hydroxyapatite (SiHA) and decorated with vascular endothelial growth factor (VEGF) have been evaluated in vitro and in vivo. Ti6Al4V-ELI scaffolds were prepared by electron beam melting and subsequently coated with Ca10(PO4)5.6(SiO4)0.4(OH)1.6 following a dip coating method. In vitro studies demonstrated that SiHA stimulates the proliferation of MC3T3-E1 pre-osteoblastic cells, whereas the adsorption of VEGF stimulates the proliferation of EC2 mature endothelial cells. In vivo studies were carried out in an osteoporotic sheep model, evidencing that only the simultaneous presence of both components led to a significant increase of new tissue formation in osteoporotic bone., 39 pages, 8 figures

Mesoporous nanospheres in the system SiO

Calcium phosphate (CaP) based hybrid materials have been synthesized through a precipitation method in aqueous medium in the presence of the anionic surfactant sodium dodecylbenzene sulfonate (SDBS) as structure directing agent. Parameters of synthesis, such as Ca/P molar ratio, surfactant concentration and initial pH, have been investigated trying to get mesostructured CaP phases. A lamellar-like hybrid mesophase consisting in several wavy layers of apatite or apatite-brushite nanoplates was successfully obtained. Materials have been characterized by several physico- chemical techniques. A potential application of these lamellar calcium phosphate based hybrids can be as matrixes in drug delivery systems with the possibility of loading hydrophobic drugs in the organic interlayers. To evaluate their potential as biomaterials, the biocompatibility of two hybrids has been studied in vitro with human Saos-2 osteoblasts. Cell assays showed that, upon the appropriate synthesis and stabilization conditions, a biocompatible CaP and SDBS mesolamellar hybrid can be prepared.

Hierarchically structured porous scaffolds based on nanocrystalline carbonated hydroxyapatite reinforced hydrogels (Gellan or Agarose) have been tested as protein release matrices while evaluation their in vitro biocompatibility. The shaping method used develops under mild conditions thus allowing the incorporation of labile substances. The Bovine Serum Albumin (BSA), employed as a model protein, has been included by using two drug-inclusion strategies: during the scaffolds preparation (in situ process) or by injection of an aqueous protein solution within (ex situ process). The release studies showed a more controlled BSA delivery when the protein was incorporated during the scaffold preparation when compared to that where the protein has been loaded in a second step (ex situ process). The release patterns can also be tailored as a function of the scaffold composition (ceramic/polysaccharide ratio and nature) as well as the drying technology employed. Biocompatibility studies demonstrated that these scaffolds, regardless of the composition, allow the culture of osteoblasts on and around the material, thus supporting the potential use of these biomaterials for bone tissue engineering.

Graphene oxide (GO) is considered to be a promising nanomaterial for biomedical applications due to its small two-dimensional shape besides its electrical and mechanical properties. However, only a few data concerning the cell responses to this material have been described and the GO biocompatibility has not been yet fully assessed. In the present study, graphene oxide nanosheets (GOs) decorated with 1-arm (1-GOs) and 6-arm (6-GOs) poly(ethylene glycol-amine) (PEG) have been incubated with cultured Saos-2 osteoblasts, MC3T3-E1 preosteoblasts and RAW-264.7 macrophages to analyze several key cell markers for in vitro biocompatibility evaluation. The results demonstrate that, after internalization, GO nanosheets are localized on F-actin filaments inducing cell-cycle alterations, apoptosis and oxidative stress in these cell types. The observed GOs effects must be considered in further studies focused on photothermal cancer therapy as a synergistic factor.

Diabetes mellitus (DM) induces bone deterioration, while mechanical stimulation promotes osteocyte-driven bone formation. We aimed to evaluate the interaction of acute exposure (24 h) to high glucose (HG) with both the pro-survival effect conferred to osteocytic MLO-Y4 cells and osteoblastic MC3T3-E1 cells by mechanical stimulation and the interaction of these cells with osteoclast precursor RAW264.7 cells. We found that 24 h of HG (25 mM) pre-exposure prevented both cell survival and ERK and β-catenin nuclear translocation upon mechanical stimulation by fluid flow (FF) (10 min) in both MLO-Y4 and MC3T3-E1 cells. However, migration of RAW 264.7 cells was inhibited by MLO-Y4 cell-conditioned medium (CM), but not by MC3T3-E1 cell-CM, with HG or FF. This inhibitory effect was associated with consistent changes in VEGF, RANTES, MIP-1α, MIP-1β MCP-1, and GM-CSF in MLO-Y4 cell-CM. RAW264.7 proliferation was inhibited by MLO-Y4 CM under static or HG conditions, but it increased by FF-CM with or without HG. In addition, both FF and HG abrogated the capacity of RAW 264.7 cells to differentiate into osteoclasts, but in a different manner. Thus, HG-CM in static condition allowed formation of osteoclast-like cells, which were unable to resorb hydroxyapatite. In contrast, FF-CM prevented osteoclastogenesis even in HG condition. Moreover, HG did not affect basal RANKL or IL-6 secretion or their inhibition induced by FF in MLO-Y4 cells. In conclusion, this in vitro study demonstrates that HG exerts disparate effects on osteocyte mechanotransduction, and provides a novel mechanism by which DM disturbs skeletal metabolism through altered osteocyte-osteoclast communication.

Vascular endothelial growth factor (VEGF) plays an essential role in angiogenesis and vascular homeostasis. Endothelial progenitor cells (EPCs) are primitive bone marrow cells participating in neovascularization and revascularization processes, which also promote bone regeneration. Synthetic hydroxyapatite (HA) has been widely used in bone repair and implant coatings. In HA-based materials, small levels of ionic substitution by silicon (Si) have significant effects on osteoclastic and osteoblastic responses. Moreover, nanocrystalline hydroxyapatites (nano-HA) display enhanced bioreactivity and beneficial effects in bone formation. In this work, the angiogenic potential of VEGF-121 adsorbed on crystalline and nanocrystalline HAs with different Si proportion is evaluated with endothelial-like cells derived from EPCs cultured on nano-HA, nano-SiHA0.25, nano-SiHA0.4, HA, SiHA0.25 and SiHA0.4 disks. The Si amount incorporated for x ¼ 0.25 is enough to yield changes in the textural parameters and surface charge without decomposing the HA phase. Si substitution for x ¼ 0.4 does not result in pure Si-substituted apatites. Si probably remains at the grain boundaries as amorphous silica in nano-SiHA0.4 and SiHA0.4 is decomposed in a-TCP and HA after 1150 �C treatment. Immobilized VEGF on nano-HA, nano-SiHA0.25, nano-SiHA0.4, HA, SiHA0.25 and SiHA0.4 maintains its function exerting a local regulation of the cell response. The crystallite size and topography of nanocrystalline HAs could produce insufficient and weak contacts with endothelial-like cells triggering anoikis. Concerning Si proportion, the best results are obtained with SiHA0.25/VEGF and nano- SiHA0.25/VEGF disks. All these results suggest the potential utility of SiHA0.25/VEGF and nano-SiHA0.25/VEGF for bone repair and tissue engineering by promoting angiogenesis.

Therapeutic strategies for bone regeneration involve the selection of suitable biomaterials, growth factors, and cell types to mimic the cellular microenvironment where molecular and mechanical signals control the reconstruction of bone tissue. The immobilization of basic fibroblast growth factor (FGF-2) on powdered silicon-substituted hydroxyapatite (Si-HA) allows to prepare a biofunctional biomaterial able to interact with bone cells in a very specific way. The biological activity of FGF-2/Si-HA, evaluated in Saos-2 osteoblasts and MC3T3-E1 preosteoblasts through the PLCγ and MAPK/ERK signal transduction pathways, shows that FGF-2 immobilized on Si-HA provides the right signals to cells stimulating crucial intracellular mechanisms of osteoblast proliferation and differentiation.

The increasing prevalence and high sanitary costs of lesions affecting the central nervous system (CNS) at the spinal cord are encouraging experts in different fields to explore new avenues for neural repair. In this context, graphene and its derivatives are attracting significant attention, although their toxicity and performance in the CNS in vivo remains unclear. Here, the subacute tissue response to 3D flexible and porous scaffolds composed of partially reduced graphene oxide is investigated when implanted in the injured rat spinal cord. The interest of these structures as potentially useful platforms for CNS regeneration mainly relies on their mechanical compliance with neural tissues, adequate biocompatibility with neural cells in vitro and versatility to carry topographical and biological guidance cues. Early tissue responses are thoroughly investigated locally (spinal cord at C6 level) and in the major organs (i.e., kidney, liver, lung, and spleen). The absence of local and systemic toxic responses, along with the positive signs found at the lesion site (e.g., filler effect, soft interface for no additional scaring, preservation of cell populations at the perilesional area, presence of M2 macrophages), encourages further investigation of these materials as promising components of more efficient material-based platforms for CNS repair., This work was supported by the Instituto de Salud Carlos III (ISCIII) and Ministerio de Economía y Competitividad (MINECO) (Grant CP13/00060), co‐financed by FEDER funding, and MINECO (Grants MAT2011‐25329 and MAT2013‐43229‐R). AGM and MCS acknowledge ISCIII‐MINECO for respective contracts associated to project CP13/00060.

Hydroxyapatite (HA) is a calcium phosphate bioceramic widely used for bone grafting and augmentation purposes. The biological response of HA can be improved through chemical and microstructural modifications, as well as by manufacturing it as macroporous implants. In the present study, calcium deficient hydroxyapatite (CDHA) and Si substituted hydroxyapatite (SiHA) macroporous scaffolds have been prepared by robocasting. In order to obtain different microstructural properties, the scaffolds have been treated at 700°C and 1250°C. The scaffolds have been characterized and tested as supports for both osteoblast growth and pre-osteoblast differentiation, as fundamental requisite for their potential use in bone tissue engineering. Morphology, viability, adhesion, proliferation, cell cycle, apoptosis, intracellular content of reactive oxygen species and interleukin-6 production were evaluated after contact of osteoblasts-like cells with CDHA and SiHA materials. An adequate interaction of osteoblasts-like cells and preosteoblasts-like cells with all these scaffolds was observed. However, the higher bone cell proliferation and differentiation on CDHA and SiHA scaffolds treated at 1250°C and the lower adsorption of albumin and fibrinogen on these materials in comparison to those treated at 700°C, suggest a better tissue response to CDHA and SiHA materials treated at high temperature.

10 páginas, 8 figuras -- PAGS nros. 2439-2448, The role of membrane cofactor protein (MCP, CD46) on human T cell activation has been analyzed. Coligation of CD3 and CD46 in the presence of PMA or CD28 costimuli enhanced IL-2, IFN-γ, or IL-10 secretion by CD4+ T lymphocytes. The effect of CD46 on IL-10 secretion did not require additional costimuli like anti-CD28 antibodies or phorbol esters. CD46 also enhanced IL-2 or IFN-γ secretion by CD4+ blasts. In contrast, IL-5 secretion was inhibited upon CD46-CD3 coligation, in all the cells analyzed. These effects were independent of IL-12 and suggest that CD46 costimulation promotes a Th1-biased response in human CD4+ T lymphocytes. CD46 enhanced TCR/CD3-induced tyrosine phosphorylation of CD3ζ and ZAP-70, as well as the activation of the ERK, JNK, and p38, but did not modify intracellular calcium. The effect of specific inhibitors shows that enhanced ERK activation contributes to augmented IFN-γ and lower IL-5 secretion and, consequently, to the Th1 bias. Cross-linking CD46 alone induced weak tyrosine phosphorylation of CD3ζ and ZAP-70. However, CD46 cross-linking by itself did not induce cell proliferation or lymphokine secretion, and pretreatment of CD4+ T lymphocytes with anti-CD46 antibodies did not significantly alter TCR/CD3 activation, This work was supported by Grants FIS 98/0037-CO2–02 from “Fondo de Investigación Sanitaria” and ISCIII-01/30 (Ministerio de Sanidad y Consumo, Spain) and Grant BMC2001–2177 from Ministerio de Ciencia y Tecnología, Spain. A.S. was recipient of a Predoctoral Fellowship of Comunidad Autónoma de Madrid

In the present study, the effects of nanocrystalline hydroxyapatite (nano-HA) and nanocrystalline Si-substituted hydroxyapatite (nano-SiHA) on osteoclast differentiation and resorptive activity have been evaluated in vitro using osteoclast-like cells. The action of these materials on proinflammatory and reparative macrophage populations was also studied. Nano-SiHA disks delayed the osteoclast differentiation and decreased the resorptive activity of these cells on their surface, as compared to nano-HA samples, without affecting cell viability. Powdered nano-SiHA also induced an increase of the reparative macrophage population. These results along with the beneficial effects on osteoblasts previously observed with powdered nano-SiHA suggest the potential of this biomaterial for modulating the fundamental processes of bone formation and turnover, preventing bone resorption and enhancing bone formation at implantation sites in treatment of osteoporotic bone and in bone repair and regeneration.

Graphene oxide (GO) has attracted the scientific community attention due to its novel properties and wide range of potential applications including hyperthermia cancer therapy. However, little is known about the GO effects on the immune function which involves both innate and adaptive defence mechanisms through the activation of different cell populations and secretion of several cytokines. The effect of different GO nanosheets designed for hyperthermia cancer therapy on macrophage and lymphocyte function should be determined before using GO for this application. Experiments The effects of GO nanosheets with 1 (1-GOs) and 6 arms (6-GOs) of polyethylene glycol on RAW-264.7 macrophages and primary splenocytes (as approximation to the in vivo situation) were evaluated through the proinflammatory cytokine secretion and the modulation of cell proliferation in the presence of specific stimuli for either T-lymphocytes (concanavalin A, anti-CD3 antibody) or B-lymphocytes/macrophages (lipopolysaccharide). Findings 6-GOs significantly increased the secretion of TNF-α by RAW-264.7 macrophages without alteration of IL-6 and IL-1β levels. The treatment of primary splenocytes with 1-GOs and 6-GOs in the presence of concanavalin A, anti-CD3 antibody and lipopolysaccharide, produced significant dose-dependent decreases of cell proliferation and IL-6 levels, revealing weak inflammatory properties of GOs which are favourable for hyperthermia cancer therapy.

Current models of the TCR / CD3 complex assume that, in mature peripheral T lymphocytes, variability is restricted to the alpha beta (or gamma delta) chains of the TCR heterodimer responsible for antigen recognition, whereas the CD3 polypeptides involved in signal transmission are invariant. Here we show that mouse CD4(+) T lymphocytes and T cell lines are bound with different avidity by anti-CD3 monoclonal antibodies. These findings cannot be accounted for by allelic differences between CD3 chains, by the nature of the TCR chains, or by the ratio of CD3epsilon delta to CD3epsilon gamma chain pairing. Rather, they are linked to heterogeneity of the N-terminal region of CD3epsilon chains, as detected by peptide-specific antibodies. In turn, these differences among CD3epsilon chains correlate with variations in the strength of TCR / CD3 interaction. N-terminal CD3epsilon heterogeneity is not due to alternative splicing mechanisms, but rather involves digestion by metalloproteases, as suggested by reverse transcription-PCR amplification and by the effect of protease inhibitors, respectively. Based on these data, we propose a model linking CD3epsilon N-terminal variability with altered CD3 recognition by monoclonal antibodies and TCR / CD3 interaction. This model suggests the possibility of distinct spatial arrangements of the TCR / CD3 complex.

CD4 cross-linking by HIV gp120 triggers CD4+ T cell death. Several authors have suggested that this effect is mediated by CD95, but this possibility is debated by other authors. In a previous work, we found by co-capping that gp120(451) and gp120MN, but not gp120(IIIB), induce lateral association of CD4 with CD95 on the T cell surface. In this work, we used fluorescence resonance energy transfer to confirm that CD4/CD95 lateral association is induced by gp120(451), but not gp120(IIIB). Moreover, we found that gp120 ability to induce the CD4/CD95 association correlates with ability to induce cell death, since gp120(451) and gp120MN induced higher levels of cell death than did gp120(IIIB) in PHA-derived CD4+ T cell lines. CD95 involvement in gp120-induced cell death was confirmed by showing that gp120(451) and gp120MN did not induce death in CD4+ T cells derived from patients with autoimmune/lymphoproliferative disease (ALD) and decreased CD95 function. Cell death induced by gp120MN was inhibited by a recombinant CD95/IgG.Fc molecule blocking the CD95/CD95L interaction. However, inhibition was late and only partial. These data suggest that the gp120-induced CD4/CD95 association exerts a dual effect: an early effect that is independent of CD95L and may be due to direct triggering of CD95 by gp120, and a late effect that may be due to sensitization of CD95 to triggering by CD95L. In line with the former effect, cell treatment with gp120MN activated caspase 3 in the presence of Fas/IgG.Fc, which shows that cell death induced by gp120MN independently of CD95L uses the same pathway as CD95.

CD44 is a family of mucin-like membrane proteins generated by alternative splicing of several exons, and participate in T cell adhesion and activation. CD44-mediated signaling involves activation of p56(lck) and leads to ZAP-70 phosphorylation. The aim of the present study was to identify the signaling pathways that follow CD44-triggered ZAP-70 phosphorylation and the molecular mechanisms underlying the CD44 interaction with p56(lck). We found that CD44 cross-linking by mAb in CD4(+) peripheral blood T cells promotes formation of a trimeric complex of Grb2, phospholipase (PLC)-gamma1 and a 36-38 kDa phosphoprotein, and the activation of PLC-gamma1. The amount of inositol triphosphate and the time kinetics of its generation were comparable to those following CD3 cross-linking. Co-capping, co-immunoprecipitation and fluorescence resonance energy transfer experiments showed that CD44 associates with CD4 and CD3 on the cell surface. This association suggests functional interplay between the CD4-TCR complex and CD44. In line with this possibility, we found that CD4 triggering by gp120, a natural ligand of CD4, potentiates CD44-mediated adhesion to hyaluronic acid. Moreover, Ca2+ mobilization induced by CD44 cross-linking by mAb was higher in a subclone of the HUT78 cell line expressing CD4 than in a non-expressing subclone.

This work extends our previous finding that lymphocyte treatment with gp120IIIB specifically induces CD4 association with several surface molecules to other molecules and to three other gp120s from different HIV-1 strains. The ability to induce this association was displayed by the four gp120s employed, i.e. gp120IIIB, gp120SF2, gp120MN and gp120(451), and the association patterns were different, as shown by both co-capping and immunoprecipitation. Co-capping showed that all four gp120s significantly potentiated CD4 association with CD3, CD45RA, CD45RB, CD38, CD26, CD59 and class I MHC molecules. By contrast, CD4 association with CD95 was induced only by gp120(451) and gp120MN; that with CD11a only by gp120SF2 and gp120MN; and that with CD27 and CD45RO only by gp120MN and gp120(451) respectively. All gp120s induced significant CD4 association with CD49d, but gp120SF2 displayed a significantly weaker effect than gp120IIIB. Induction of association was not mediated by inside-out signaling via the CD4-associated tyrosine kinase p58lck, since it was not inhibited by the tyrosine kinase inhibitors herbymicin and genistein, nor by CD45 bridging between CD4 and the associating molecule, since similar patterns of association were detected IN cells expressing different CD45 isoform patterns. Moreover, it was not mediated by chemokine receptors interacting with the gp120 V3 loop, since RANTES did not alter the gp120-induced CD4 association pattern. By contrast, the observation that gp120s from four HIV-1 strains induce different CD4 association patterns suggests that gp120 directly interacts with the associating molecules, possibly via their hypervariable regions.

Hypothesis: Synthetic hydroxyapatite (HA) and Si substituted hydroxyapatite (SiHA) are calcium phosphate ceramics currently used in the field of dentistry and orthopaedic surgery. The preparation of both biomaterials as polycrystalline solid pieces or grains formed by nanocrystallites has awakened a great interest to enhance the bioactive behavior due to the microstructural defects and the higher surface area. The study of the macrophage and lymphocyte behavior in contact with nanocrystalline HA and SiHA will allow to elucidate the immune response which conditions the success or rejection of these biomaterials. Experiments: HA and SiHA granules (with sizes of tens of microns) have been prepared by controlled aqueous precipitation avoiding subsequent high temperature sintering. HA and SiHA granules were constituted by crystallites smaller than 50 nm. The effects of both nanocrystalline materials on immune system have been evaluated with macrophages (main components of innate immune system) and T lymphocytes (specific cells of adaptive response) after short-term culture as in vitro models of the early immune response. Findings: Significant decreases of macrophage proliferation and phagocytic activity, increased production of inflammatory cytokines (IL-6, TNF-a) and T lymphocyte apoptosis, were induced by these nanocrystalline ceramics suggesting that, after in vivo implantation, they induce significant effects on immune responses, including an early activation of the innate immune system. (C) 2013 Elsevier Inc. All rights reserved.

Graphene and more specifically, nanographene oxide (GO) has been proposed as a highly efficient antitumoral therapy agent. Nevertheless, its cell uptake kinetics, its influence in different types of cells and the possibility of controlling cellular internalization timing, is still a field that remains unexplored. Herein, different cell types have been cultured in vitro for several incubation periods in the presence of 0.075 mg ml(-1) pegylated GO solutions. GO uptake kinetics revealed differences in the agent's uptake amount and speed as a function of the type of cell involved. Osteoblast-like cells GO uptake is higher and faster without resulting in greater cell membrane damage. Moreover, the dependence on the commonly used PEG nature (number of branches) also influences the viability and cell uptake speed. These facts play an important role in the future definition of timing parameters and selective cell uptake control in order to achieve an effective therapy.

Cancer patients are often treated with biological response modifiers to enhance immunological functions. However, little is known about the actual mechanism of action of many of these substances. Therefore, we were interested in the effect of i.p. treatment with porcine low-molecular-weight spleen peptides, which are used during supportive cancer therapy, on lymphoid cell populations and function in mice. After treatment with 0.5 microgram peptides/kg body weight for 14 consecutive days, lymphokine secretion and the generation of cytotoxic T-cells were significantly enhanced as compared with controls. However, there was no effect on the number of cells or the percentage of cells expressing functional surface markers in secondary lymphoid organs.

Si-doped hydroxyapatite (Si-HA) is a suitable ceramic for the controlled release of agents to improve bone repair. We recently showed that parathyroid hormone-related protein (PTHrP) (107-111) (osteostatin) has remarkable osteogenic features in various in vitro and in vivo systems. Fibroblast growth factor (FGF)-2 modulates osteoblastic function and induces angiogenesis, and can promote osteoblast adhesion and proliferation after immobilization on Si-HA. In the present study we examined whether osteostatin might improve the biological efficacy of FGF-2-coated Si-HA in osteoblastic MC3T3-E1 cells in vitro. We found that Si-HA/FGF-2 in the presence or absence of osteostatin (100 nM) similarly increased cell growth (by about 50%). However, addition of the latter peptide to Si-HA/FGF-2 significantly enhanced gene expression of Runx2, osteocalcin, vascular endothelial growth factor (VEGF) and the VEGF receptors 1 and 2, without significantly affecting that of FGF receptors in these cells. Moreover, secreted VEGF in the MC3T3-E1 cell conditioned medium, which induced the proliferation of pig endothelial-like cells, was also enhanced by these combined factors. The synergistic action of osteostatin and Si-HA/FGF-2 on the VEGF system was abrogated by a mitogen-activated protein kinase inhibitor (U0126) and by the calcium antagonist verapamil. This action was related to an enhancement of alkaline phosphatase activity and matrix mineralization in MC3T3-E1 cells, and also in primary human osteoblastic cells. These in vitro data show that osteostatin increases the osteogenic efficacy of a Si-HA/FGF-2 biomaterial by a mechanism involving mitogen-activated protein kinases and intracellular Ca(2+). These findings provide an attractive strategy for bone tissue engineering.

The use of biomaterials as implantable thermoseeds under the action of an external magnetic field is a very interesting methodology to focus the heat into the target tumors as osteosarcoma. In this study, biocompatible and bioactive G15GC85 thermoseeds, tailored through the combination of sol–gel glasses (G) with a magnetic glass ceramic (GC), were used to induce hyperthermia on cultured human osteosarcoma cells after exposition to alternating magnetic field (MF, 100 kHz/200 Oe). G15GC85 magnetic glass–glass ceramic thermoseeds induced in vitro effective hyperthermia with drastic reduction in proliferation of human osteosarcoma Saos-2 cells and high increase of apoptotic cells after two 40 min consecutive sessions of MF. Deep cell morphology alterations were observed after this hyperthermic treatment, and the proteomic analysis revealed modification of gamma actin molecular properties related to cytoskeleton alterations. These results indicate that G15GC85 thermoseeds allow to induce in vitro effective hyperthermia on human osteosarcoma cells. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

8 páginas, 3 figuras, 2 tablas -- PAGS nros. 8-15, CD3ɛ chains are essential to the structure, expression and signaling of T cell receptors. Here, we extend to human CD3ɛ our previous data in mouse CD3ɛ showing that, in T cells, proteolytic processing of the acidic N-terminal sequence of CD3ɛ chains generate distinct polypeptide species that can be identified by two-dimension (IEF-SDS PAGE) electrophoresis and immunoblot. This was shown first by showing the processing of a fusion protein of GFP and the extracellular domain of mouse CD3ɛ (mCD3GFP) expressed in Jurkat cells. Secondly, pI heterogeneity was also found in human CD3ɛ chains immunoprecipitated from the surface of Jurkat cells or PHA blasts of human blood T lymphocytes. Comparison of CD3ɛ chains from 27 different species shows that their N-terminal sequences share a strong acidic nature, despite the large differences in terms of length and composition, even among closely related species. Our results suggest that generation of CD3ɛ chain isoforms with different N-terminal sequence and pI is a general phenomenon. Thus, as previously observed in the mouse, the relative abundance of CD3ɛ chain species might regulate TCR/CD3 structure and function, including the strength of the interactions between CD3 dimers and the TCR clonotypic receptors, as well as TCR/CD3 activation thresholds. Interestingly, CD3ɛ chains from 7 out of 27 species studied have putative N-glycosylation (NxS or NxT) motifs in their Ig extracellular domain. Their location, plus the conservation of residues involved in domain organization, the interactions with other CD3 chains, or the TCR, and signal triggering add new data useful to establish a permissive topology for the interaction between CD3 dimers and the TCR chains, Funded by grants from Ministerio de Sanidad y Consumo, Spain (ISCIII-05/054, PI070620, PI070484); Ministerio de Ciencia y Tecnología, Spain (SAF2004-06852). R.B. was recipient of a predoctoral fellowship of Ministerio de Ciencia y Tecnología, Spain. P.P. is a Tenured Scientist of the Consejo Superior de Investigaciones Científicas at the Centro Nacional de Microbiología, Instituto de Salud Carlos III

12 p.-6 fig., The antigen T cell receptor (TCR)4 complex is responsible for antigen recognition through TCRαβ (or TCRγδ) variable heterodimers. These are noncovalently associated with CD3γ, CD3δ, CD3ϵ, and ζ (CD247) polypeptides involved in the initiation of signal transduction and the control of complex expression (reviewed in Refs. 1 and 2). Current structural models of the TCR-CD3 complex support the idea that each minimal subunit contains one TCRαβ (or TCRγδ) heterodimer and two CD3ϵ polypeptides per TCR heterodimer (3–8). CD3ϵ ectodomains pair noncovalently with one CD3γ or CD3δ chain through unique excluding sites in their G strands and membrane-proximal stalk sequences, and it is thus assumed that there is one CD3ϵδ and one CD3ϵγ dimer per complex (9–14). Finally, it has been shown that covalently linked ζ chain homodimers are needed for efficient transport of complete TCR-CD3 complexes from the endoplasmic reticulum to the cell surface (reviewed in Ref. 2), and data from in vitro assembly of TCR-CD3 chains suggest the association of one ζ dimer per complex (3, 4). From these data, it follows that the minimal TCR-CD3 complex unit contains eight polypeptides (αβ·ϵδ·ϵγ·ζ2). The data summarized above have been generated using many different cells and cell lines in diverse experimental approaches, and it would be reasonable to conclude that the TCR-CD3 complex is a constant structure, the components of which (beyond those differences arising from V region variability) are equal in all T cells. However, the existing data also indicate that there are profound quantitative and qualitative changes during the development of not only T lymphocytes (15), but also among different mature T cell subsets or T cell lines. Concerning CD3, different ratios of CD3γ and CD3δ polypeptides in TCR-CD3 complexes from different T cell lines have been reported (9). Furthermore, CD3δ chains are absent, and there are wide differences in CD3γ chain glycosylation in γδ T cells (16). Even so, TCRγδ-CD3 complexes contain two CD3ϵγ dimers per complex (8). TCR-CD3 complexes naturally form aggregates, the degree of aggregation of which within one cell or among T cells cannot be easily ascribed to the nature of the TCR antigen recognition unit (7, 17). Differences in TCR-CD3 structure among human or mouse T cell lines have been also detected biochemically and by differences in their relative recognition by anti-TCR or anti-CD3 antibodies (18–20)., All these differences might have important functional consequences if they can alter the factors determining the efficiency of TCR-mediated signals. These include the efficiency of spontaneous (7, 17) or ligand-induced oligomerization/polymerization of TCR-CD3 complexes, the sensitivity to induction of conformational changes upon ligand binding (21), and the efficiency of coreceptor association with the TCR-CD3 complex (22, 23). In turn, these differences could correlate with the amount and distribution of molecules involved in the activation of distinct pathways and/or the threshold and kinetic patterns of T cell activation. In a previous study (18), we detected differences in the recognition of mouse CD3 by monoclonal antibodies that were linked to differences in the N-terminal sequence of CD3ϵ as determined by recognition with an N-terminal peptide-specific antibody. These differences were not due to alternative splicing of the mini-exons coding for the N-terminal sequence of CD3ϵ, but were due to degradation by proteinases (including metalloproteinases) sensitive to phenanthroline. Interestingly, the association of CD3 with the TCR is weaker when CD3ϵ N-terminal sequences are degraded (18). Here, we have taken advantage of the presence of negatively charged amino acid residues in the N-terminal sequence of CD3ϵ to further analyze this phenomenon. Removal of these charged residues should affect the isoelectric point of CD3ϵ, rendering isoforms with distinct pI values that could be distinguished by two-dimensional PAGE. Furthermore, we have generated mutant CD3ϵ chains lacking charged amino acids and showed that their loss can enhance the response to TCR-mediated activation, This work was supported in part by Grants ISCIII-01/30 and ISCIII-05/054 from the Ministerio de Sanidad y Consumo (Spain) and Grants BMC2001-2177 and SAF2004-06852 from the Ministerio de Ciencia y Tecnología (Spain) (to P. P. and J. M. R.). The costs of publication of this article were defrayed in part by the payment of page charges

9 páginas, 5 figuras, 1 tabla -- PAGS nros. 3571-3579, The complement regulatory protein CD46 (MCP, membrane cofactor protein) is used as a cell receptor by a number of bacterial and viral pathogens, including Streptococcus pyogenes (Group A Streptococci). The highly variable M (Emm) proteins are virulence factors of S. pyogenes, and Emm proteins of serotypes 5, 6 or 22 are able of binding to CD46, thus mediating the binding of Streptococci to human cells. In this work, using a soluble construction encompassing the extracellular domain of human CD46, we have analyzed its binding to clinical isolates of S. pyogenes, including isolates of the M types 1, 3 and 18 that are frequently found in invasive infections or rheumatic fever. Our data show a strong binding of CD46 to bacteria of M types 1, 3, 8, 18, 24, 28, 29, 31 and 78; weak binding to M6 and M29 and no binding to M types 11, 12, M27 or M30. Surprisingly, CD46 bound to isogenic mutants of one clinical M18 isolate lacking the Emm protein or Emm and the Emm-related protein Enn, regardless of having capsule or not. In addition, these isogenic mutants bound to keratinocytes in a CD46-dependent manner, confirming the role of CD46 as one of the cell receptors for Group A Streptococci. Furthermore, CD46 did not bind to a recombinant Emm 18 construct, confirming that Emm is not involved in CD46 binding to M18 bacteria. Emm-dependent and -independent CD46 binding of clinical isolates of Streptococci confirms the importance of CD46 as a cell target that might confer pathogens some biological advantages over the host., Financial support was provided by grants SAF2004-06852 (Ministerio de Ciencia y Tecnología, Spain) and ISCIII-05/054 (Ministerio de Sanidad y Consumo, Spain). A. Sánchez was recipient of a predoctoral fellowship from Comunidad Autónoma de Madrid. M.A. Oliver was supported by Instituto de Salud Carlos III through a fellowship from Red Respira (RTIC C03/11)

Escherichia coli is a common urinary pathogen whose uptake into epithelial cells is mediated by attachment through type 1 fimbriae. In this study, we show by using using human urinary tract epithelial cells that maximal internalization of E. coli is achieved only when bacteria are opsonized with complement. The concentrations of complement proteins in the urine rise sufficiently during infection to allow bacterial opsonization. The complement regulatory protein, CD46 (membrane cofactor protein), acts in cohort with fimbrial adhesion to promote the uptake of pathogenic E. coli. This uptake is inhibited by RNA interference to lower the expression of CD46 and by soluble CD46 that will competitively inhibit opsonized bacteria binding to cell surface CD46. We propose that efficient internalization of uropathogenic E. coli by the human urinary tract depends on cooperation between fimbrial-mediated adhesion and C3 receptor (CD46)–ligand interaction. Complement receptor–ligand interaction could pose a new target for interrupting the cycle of reinfection due to intracellular bacteria.

H4/ICOS is a costimulatory molecule related to CD28. Its effects on early TCR signals have been analyzed in mouse CD4(+) Th2 cells, expressing H4/ICOS at higher levels than Th1 clones. Anti-H4/ICOS antibodies strongly enhanced CD3-mediated tyrosine phosphorylation of ZAP-70, zeta, or Vav, as well as extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38 MAP kinase activation in these cells. The association of phosphoinositide 3-kinase (PI-3K) to H4/ICOS was enhanced by H4/ICOS cross-linking, and PI-3K inhibitors inhibited ERK and JNK activation and IL-4/IL-10 secretion, but not p38 MAP kinase or ZAP-70 activation. H4/ICOS-mediated activation of JNK, but not ERK or p38, is partially dependent on the expression of CD4 by the cells, whereas H4/ICOS costimulation is partially independent on CD28 expression. Cytochalasin D, an inhibitor of actin polymerization, inhibited ZAP-70, MAP kinase activation, or IL-4/IL-10 secretion. Neither cyclosporin A nor inhibitors of PKC produced detectable inhibition of ZAP-70 phosphorylation or MAP kinase activation in these Th2 cells. Cyclosporin A strongly inhibited IL-4, but not IL-10 secretion. ERK or JNKinhibitors partially inhibited IL-4 and IL-10 secretion, while PKC or p38 inhibitors had no significant effects on IL-4 or IL-10 secretion. Taken together, our data show clear similarities of costimulation mechanisms between H4/ICOS and CD28 during the early steps of TCR activation.