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Focus on advancement of energy storage has now turned to curbing carbon emissions in the transportation sector by adopting electric vehicles (EVs). Technological advancements in lithium-ion batteries (LIBs), valued for their lightweight and high capacity, are critical to making this switch a reality. Integrating structurally enhanced LIBs directly into vehicular design tackles two EV limitations: vehicle range and weight. In this study, 3D-carbon (3D-C) lattices, prepared with an inexpensive stereolithography-type 3D printer followed by carbonization, are proposed as scaffolds for Li metal anodes for structural LIBs. Mechanical stability tests revealed that the 3D-C lattice can withstand a maximum stress of 5.15 ± 0.15 MPa, which makes 3D-C lattices an ideal candidate for structural battery electrodes. Symmetric cell tests show the superior cycling stability of 3D-C scaffolds compared to conventional bare Cu foil current collectors. When 3D-C scaffolds are used, a small overpotential (≈0.075 V) is retained over 100 cycles at 1 mA cm-2 for 3 mAh cm-2, while the overpotential of a bare Cu symmetric cell is unstable and increased to 0.74 V at the 96th cycle. The precisely oriented internal pores of the 3D-C lattice confine lithium metal deposits within the 3D scaffold, effectively preventing short circuits.
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Silicon has gained significant attention as a lithium-ion battery anode material due to its high theoretical capacity compared to conventional graphite. Unfortunately, silicon anodes suffer from poor cycling performance caused by their extreme volume change during lithiation and de-lithiation. Compositing silicon particles with 2D carbon materials, such as graphene, can help mitigate this problem. However, an unaddressed challenge remains: a simple, inexpensive synthesis of Si/graphene composites. Here, a one-step laser-scribing method is proposed as a straightforward, rapid (≈3 min), scalable, and less-energy-consuming (≈5 W for a few minutes under air) process to prepare Si/laser-scribed graphene (LSG) composites. In this research, two types of Si particles, Si nanoparticles (SiNPs) and Si microparticles (SiMPs), are used. The rate performance is improved after laser scribing: SiNP/LSG retains 827.6 mAh g-1 at 2.0 A gSi+C -1, while SiNP/GO (before laser scribing) retains only 463.8 mAh g-1. This can be attributed to the fast ion transport within the well-exfoliated 3D graphene network formed by laser scribing. The cyclability is also improved: SiNP/LSG retains 88.3% capacity after 100 cycles at 2.0 A gSi+C -1, while SiNP/GO retains only 57.0%. The same trend is found for SiMPs: the SiMP/LSG shows better rate and cycling performance than SiMP/GO composites.
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We report on the efficient collimation of soft X-rays with an energy of 277 eV by a halved polycapillary lens (PCL), made of borosilicate glass. Using electron-excited, micro fluorescence emission in the focus of the PCL, experiments reveal an angular divergence of (6.9 ± 0.2)â mrad in the far field of the emitted beam. For a source of ≈5µm in size, that result is confirmed by simulations, obtained with a newly developed ray tracing code. An analytical fit model is proposed and applied to characterize the evolution of the measured as well as calculated, three-dimensional (3-D) intensity distribution. The photon flux density in a free-space propagation distance of (0.4 - 0.9)â m from the PCL is enhanced by a factor of ≈(30 - 90) in comparison to the direct, not collimated radiation, as it is detected through a mm-sized transmission slit. Our findings could help to establish the halved PCL as a versatile tool in the table-top metrology of optical elements, such as mirrors and gratings for soft X-rays.
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We correct values and figures for the resolution of the spectrometer, as proposed in [Opt. Express25, 31840 (2017)10.1364/OE.25.031840OPEXFF1094-4087]. The new results take into account previously unknown, incoherent phase fluctuations, caused by the polycapillary lens (PCL), and estimate the realistic performance of the instrument.
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Imposing additional confinement in two-dimensional (2D) materials yields further control over their electronic, optical, and topological properties. However, synthesis of ultranarrow nanoribbons (NRs) remains challenging, particularly for transition metal dichalcogenides (TMDs), and synthesizing TMD NRs narrower than 50 nm has remained elusive. Here, we report the vapor-phase synthesis of ultranarrow TaS2 NRs. The NRs are grown within carbon nanotubes, limiting their width and layer number, while stabilizing them against the environment. The NRs reach monolayer thickness and exhibit widths down to 2.5 nm. Atomic-resolution scanning transmission electron microscopy reveals the detailed atomic structure of the ultranarrow NRs and we observe a hitherto unseen atomic structure supermodulation of ordered defect arrays within the NRs. Density functional theory calculations show the presence of flat bands and boundary-localized states, and help identify the atomic configuration of the supermodulation. Nanotube-templated synthesis represents a unique, transferable, and broadly deployable route toward ultranarrow TMD NR growth.
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The structure of MX3 transition metal trichalcogenides (TMTs, with M a transition metal and X a chalcogen) is typified by one-dimensional (1D) chains weakly bound together via van der Waals interactions. This structural motif is common across a range of M and X atoms (e.g., NbSe3, HfTe3,TaS3), but not all M and X combinations are stable. We report here that three new members of the MX3 family which are not stable in bulk, specifically NbTe3, VTe3, and TiTe3, can be synthesized in the few- (2-4) to single-chain limit via nanoconfined growth within the stabilizing cavity of multiwalled carbon nanotubes. Transmission electron microscopy (TEM) and atomic-resolution scanning transmission electron microscopy (STEM) reveal the chain-like nature and the detailed atomic structure. The synthesized materials exhibit behavior unique to few-chain quasi-1D structures, such as few-chain spiraling and a trigonal antiprismatic rocking distortion in the single-chain limit. Density functional theory (DFT) calculations provide insight into the crystal structure and stability of the materials, as well as their electronic structure.
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This paper presents a robotic capture concept that was developed as part of the e.deorbit study by ESA. The defective and tumbling satellite ENVISAT was chosen as a potential target to be captured, stabilized, and subsequently de-orbited in a controlled manner. A robotic capture concept was developed that is based on a chaser satellite equipped with a seven degrees-of-freedom dexterous robotic manipulator, holding a dedicated linear two-bracket gripper. The satellite is also equipped with a clamping mechanism for achieving a stiff fixation with the grasped target, following their combined satellite-stack de-tumbling and prior to the execution of the de-orbit maneuver. Driving elements of the robotic design, operations and control are described and analyzed. These include pre and post-capture operations, the task-specific kinematics of the manipulator, the intrinsic mechanical arm flexibility and its effect on the arm's positioning accuracy, visual tracking, as well as the interaction between the manipulator controller and that of the chaser satellite. The kinematics analysis yielded robust reachability of the grasp point. The effects of intrinsic arm flexibility turned out to be noticeable but also effectively scalable through robot joint speed adaption throughout the maneuvers. During most of the critical robot arm operations, the internal robot joint torques are shown to be within the design limits. These limits are only reached for a limiting scenario of tumbling motion of ENVISAT, consisting of an initial pure spin of 5 deg/s about its unstable intermediate axis of inertia. The computer vision performance was found to be satisfactory with respect to positioning accuracy requirements. Further developments are necessary and are being pursued to meet the stringent mission-related robustness requirements. Overall, the analyses conducted in this study showed that the capture and de-orbiting of ENVISAT using the proposed robotic concept is feasible with respect to relevant mission requirements and for most of the operational scenarios considered. Future work aims at developing a combined chaser-robot system controller. This will include a visual servo to minimize the positioning errors during the contact phases of the mission (grasping and clamping). Further validation of the visual tracking in orbital lighting conditions will be pursued.
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A collimating polycapillary half lens, traditionally used in the medium and hard X-ray band, is operated at a photon energy of 36 eV for the first time. While the transmission still exceeds 50%, the measured and simulated spatial resolution and angular divergence approach 0.4 mm or less and at most 20 mrad, respectively. This unexpected, superior performance of the polycapillary optic in the extreme Ultraviolet could enable the design of an efficient, versatile and compact spectrometer for inverse photoemission spectroscopy (IPES): Its wavelength-dispersive component, a customized reflection zone plate, can maintain an energy resolution of 0.3 eV, whereas the sensitivity may be enhanced by more than one order of magnitude, compared to conventional spectrometers. Furthermore, the overall length of 0.9 m would allow for an eased alignment and evacuation. We see a significant potential for numerous polycapillary-based XUV / soft X-ray instruments in the future, in particular after further optimization for this long wavelength regime.
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Waveguide bundles in bulk glass materials, consisting of several parallel scans of refractive index modifications, have been generated with a low-repetition femtosecond laser. Additionally, Bragg grating (BG) structures for 840 and 1550 nm have been introduced by segmentation of the central scan. A spectral loss in the transmission signal of >36 dB was achieved at 1550 nm with a second-order Bragg grating waveguide (BGW) in fused silica, which corresponds to an intrinsic grating efficiency of >16 dB/cm. This is to our knowledge the strongest BG structure realized in glass with a femtosecond laser. The BGW were proven to be stable up to a temperature of 250°C in fused silica. The diameter of the waveguide bundles can be adapted very easily for a broad range of wavelengths and have been demonstrated for diameters between 1 and 50 µm. The transmission properties of the waveguide bundles are affected minorly by the insertion of BG structures, which opens the ability for adjusting the BGW for a broad range of wavelength in single-mode or multimode optical circuits. BGW have been realized successfully in fused silica, borosilicate glass (BOROFLOAT 33), and AF 32 eco Thin Glass from Schott.
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BACKGROUND: Tumors can be targeted by the adoptive transfer of chimeric antigen receptor (CAR) redirected T-cells. Antigen-specific expansion protocols are needed to generate large quantities of redirected T-cells. We aimed to establish a protocol to expand functional active NY-ESO-1-specific redirected human CD8(+) T-cells. MATERIALS AND METHODS: The anti-idiotypic Fab antibody A4 with specificity for HLA-A 0201/NY-ESO-1157-165 was tested by competition assays using a HLA-A 0201/NY-ESO-1157-165 tetramer. HLA-A 0201/NY-ESO-1157-165 redirected T-cells were generated, expanded and tested for CAR expression, cytokine release, in vitro cytolysis and protection against xenografted HLA-A 0201/NY-ESO-1157-165-positive multiple myeloma cells. RESULTS: A4 demonstrated antigen-specific binding to HLA-A 0201/NY-ESO-1157-165 redirected T-cells. Expansion with A4 resulted in 98% of HLA-A 0201/NY-ESO-1157-165 redirected T-cells. A4 induced strong proliferation, resulting in a 300-fold increase of redirected T-cells. After expansion protocols, redirected T-cells secreted Interleukin-2, (IL-2), interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) and lysed target cells in vitro and were protective in vivo. CONCLUSION: A4 expanded HLA-A 0201/NY-ESO-1157-165 redirected T-cells with preservation of antigen-specific function.
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Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas de la Membrana/inmunología , Mieloma Múltiple/terapia , Animales , Afinidad de Anticuerpos , Linfocitos T CD8-positivos/trasplante , Línea Celular Tumoral , Proliferación Celular , Sistema Libre de Células , Técnicas de Cocultivo , Citotoxicidad Inmunológica , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoterapia Adoptiva , Activación de Linfocitos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Trasplante de Neoplasias , Linfocitos T Citotóxicos/inmunología , Carga TumoralRESUMEN
Abstract Agonist-induced Ca(2+) oscillations in many cell types are triggered by Ca(2+) release from intracellular stores and driven by store-operated Ca(2+) entry. Stromal cell-interaction molecule (STIM) 1 and STIM2 serve as endoplasmic reticulum Ca(2+) sensors that, upon store depletion, activate Ca(2+) release-activated Ca(2+) channels (Orai1-3, CRACM1-3) in the plasma membrane. However, their relative roles in agonist-mediated Ca(2+) oscillations remain ambiguous. Here we report that while both STIM1 and STIM2 contribute to store-refilling during Ca(2+) oscillations in mast cells (RBL), T cells (Jurkat) and human embryonic kidney (HEK293) cells, they do so dependent on the level of store depletion. Molecular silencing of STIM2 by siRNA or inhibition by G418 suppresses store-operated Ca(2+) entry and agonist-mediated Ca(2+) oscillations at low levels of store depletion, without interfering with STIM1-mediated signals induced by full store depletion. Thus, STIM2 is preferentially activated by low-level physiological agonist concentrations that cause mild reductions in endoplasmic reticulum Ca(2+) levels. We conclude that with increasing agonist concentrations, store-operated Ca(2+) entry is mediated initially by endogenous STIM2 and incrementally by STIM1, enabling differential modulation of Ca(2+) entry over a range of agonist concentrations and levels of store depletion.
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Señalización del Calcio , Calcio/metabolismo , Moléculas de Adhesión Celular/metabolismo , Animales , Canales de Calcio/genética , Canales de Calcio/metabolismo , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Drosophila , Retículo Endoplásmico/metabolismo , Silenciador del Gen , Gentamicinas/farmacología , Células HEK293 , Humanos , Mastocitos/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , ARN Interferente Pequeño , Molécula de Interacción Estromal 1 , Molécula de Interacción Estromal 2 , Linfocitos T/metabolismoRESUMEN
OBJECTIVE: Spondylarthritides (SpA), including ankylosing spondylitis (AS), are common inflammatory rheumatic diseases that are strongly associated with positivity for the HLA class I allotype B27. HLA-B27 normally forms complexes with ß(2) -microglobulin (ß(2) m) and peptide to form heterotrimers. However, an unusual characteristic of HLA-B27 is its ability to form ß(2) m-free heavy chain homodimers (HLA-B27(2) ), which, unlike classic HLA-B27, bind to killer cell immunoglobulin-like receptor 3DL2 (KIR-3DL2). Binding of HLA-B27(2) to KIR-3DL2-positive CD4+ T and natural killer (NK) cells stimulates cell survival and modulates cytokine production. This study was undertaken to produce an antibody to HLA-B27(2) in order to confirm its expression in SpA and to inhibit its proinflammatory properties. METHODS: We generated monoclonal antibodies by screening a human phage display library positively against B27(2) and negatively against B27 heterotrimers. Specificity was tested by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) assay, and fluorescence-activated cell sorting (FACS) analysis of B27(2) -expressing cell lines and peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with SpA. Functional inhibition of KIR-3DL2-B27(2) interactions was tested using cell lines and PBMCs from patients with SpA. RESULTS: Monoclonal antibody HD6 specifically recognized recombinant HLA-B27(2) by ELISA and by SPR assay. HD6 bound to cell lines expressing B27(2) . FACS revealed binding of HD6 to PBMCs and SFMCs from patients with AS but not from controls. HD6 inhibited both the binding of HLA-B27(2) to KIR-3DL2 and the survival and proliferation of KIR-3DL2-positive NK cells. Finally, HD6 inhibited production of the proinflammatory disease-associated cytokine interleukin-17 by PBMCs from patients with AS. CONCLUSION: These results demonstrate that antibody HD6 has potential for use in both the investigation and the treatment of AS and other B27-associated spondylarthritides.
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Antígeno HLA-B27/inmunología , Inflamación/inmunología , Células Asesinas Naturales/inmunología , Espondiloartritis/inmunología , Linfocitos T/inmunología , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Línea Celular , Antígeno HLA-B27/metabolismo , Humanos , Inflamación/metabolismo , Células Asesinas Naturales/metabolismo , Espondiloartritis/metabolismo , Linfocitos T/metabolismoRESUMEN
Activation of murine glucocorticoid-induced tumor necrosis factor-related receptor (mGITR) by its natural ligand (GITRL) or antiGITR agonist mAb enhances T-cell responses, inhibits regulatory T-cell (Treg)-mediated suppression and induces tumor immunity in a variety of murine tumor models. However, systemic administration of these costimulatory agents can lead to global T-cell activation and autoimmunity. To specifically manipulate the T-cell compartment in the tumor microenvironment we propose to target the tumor infiltrating T cells with a bispecific mGITRL fusion protein. For that purpose, mGITRL is linked to a single-chain antibody targeting fibroblast activation protein (FAP) as FAP expression is restricted to cancer-associated fibroblasts (CAFs) found in the stroma of epithelial cancers. AntiFAP-mGITRL fusion protein forms dimers and binds to murine GITR with 1.2 µM affinity and to murine FAP with 4.5 nM. The construct is able to costimulate CD8+ and CD4+ effector T cells resulting in increased proliferation, IFN-γ and IL-2 production. This costimulatory effect is enhanced when the fusion protein is bound to a FAP-positive cell line mimicking FAP CAFs. In suppression assays, membrane-bound antiFAP-mGITRL is 100-fold more effective in overcoming Treg-mediated suppression than unbound fusion protein. These studies suggest that targeted tumor therapy with antiFAP-mGITRL fusion protein could induce tumor rejection while minimizing autoimmune side effects.
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Antígenos de Neoplasias/metabolismo , Inmunoterapia , Neoplasias Experimentales/inmunología , Receptores de Factor de Crecimiento Nervioso/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Animales , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citocinas/genética , Citocinas/metabolismo , Endopeptidasas , Gelatinasas/genética , Gelatinasas/inmunología , Gelatinasas/metabolismo , Proteína Relacionada con TNFR Inducida por Glucocorticoide , Activación de Linfocitos/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/tratamiento farmacológico , Estructura Terciaria de Proteína/genética , Receptores de Factor de Crecimiento Nervioso/inmunología , Receptores del Factor de Necrosis Tumoral/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/inmunología , Serina Endopeptidasas/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismo , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patologíaRESUMEN
Costimulation is a fundamental principle of T-cell activation. In addition to T-cell receptor engagement, the interaction between CD80 and/or CD86 with CD28 and/or cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptors is required to regulate T-cell activation and tolerance. While the importance of costimulation is clearly established, the exact molecular mechanism is unknown. We demonstrate that T-cell proliferation and the ability of CD8(+) T-effector cells to kill were enhanced slightly by CD80 but dramatically by CD86 costimulation. To further analyse the cellular process of costimulation, we developed a single-cell assay to analyse Ca(2+) signals following costimulation with bi-specific antibodies. We found that this stimulation method worked in every human T-cell that was analysed, making it one of the most efficient T-cell activation methods to date for primary human T cells. The enhanced proliferation and killing by costimulation was paralleled by an increase of Ca(2+) influx following CD86 costimulation and it was dependent on CD28/CTLA-4 expression. The enhanced Ca(2+) influx following CD86 costimulation was abrogated by an antibody that interfered with CD28 function. The differences in Ca(2+) influx between CD80 and CD86 costimulation were not dependent on the depletion of Ca(2+) stores but were eliminated by the application of 10 mum 2-aminoethyldiphenyl borate which has recently been shown to enhance stromal interaction molecule 2 (STIM2)-dependent Ca(2+) entry while reducing STIM1-dependent Ca(2+) entry. Our data indicate that differences in the efficiency of costimulation are linked to differences in Ca(2+) entry.
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Anticuerpos Monoclonales , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Linfocitos T CD8-positivos/metabolismo , Proteínas Recombinantes de Fusión , Antígeno B7-1/inmunología , Antígeno B7-2/inmunología , Compuestos de Boro/farmacología , Complejo CD3/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/inmunología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Proliferación Celular , Citotoxicidad Inmunológica , Humanos , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ingeniería de Proteínas , Molécula de Interacción Estromal 2RESUMEN
MHC-peptide-specific Fab antibodies binding to HLA-A*0201 complexes presenting the wild-type EAAGIGILTV (EAA) or analogue Melan-A 10-mer ELAGIGILTV (ELA) peptide were generated to study efficacy of peptide processing and presentation. None of the selected Fab antibodies detected the naturally processed EAA/HLA-A*0201 complex on melanoma tumor cells, confirming the known low peptide number on the cell surface. To study the effect of peptide presentation and processing in more detail, genes coding for the A27L-mutated Melan-A protein or the processed ELA peptide were introduced into HLA-A*0201(+) B cells by infection with the respective recombinant vaccinia virus construct producing equimolar amounts of GFP-ubiquitin directly linked to the fragment of interest. Correlating GFP expression to actual numbers of peptide presented, 1100-2600 [corrected] ELA peptides had to be synthesized to be presented by a single MHC class I antigen-peptide complex. This number increased 10- to 20-fold when ELA peptide presentation from the A27L-mutated full length Melan-A protein was studied, since 16000-52000 [corrected] GFP molecules needed to be synthesized for the detection of one ELA peptide. Our results indicate that peptide processing rather than presentation is the rate-limiting step in our experimental setting and is much more ineffective for Melan-A than has been previously shown for other MHC class I-restricted epitopes.
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Especificidad de Anticuerpos/inmunología , Presentación de Antígeno/inmunología , Antígenos de Neoplasias/metabolismo , Antígenos HLA-A/metabolismo , Fragmentos Fab de Inmunoglobulinas/inmunología , Proteínas de Neoplasias/metabolismo , Antígenos de Neoplasias/inmunología , Western Blotting , Citometría de Flujo , Antígenos HLA-A/inmunología , Antígeno HLA-A2 , Humanos , Antígeno MART-1 , Proteínas de Neoplasias/inmunología , Péptidos/inmunología , Péptidos/metabolismoRESUMEN
Major depression (MD) is underdiagnosed and undertreated in patients with temporal lobe epilepsy (TLE). Side effects of some antidepressants, like increased risk of seizures and drug-drug interactions with anticonvulsants, contribute to undertreatment of MD in patients with TLE. We analyzed post hoc the data from 2 years of treatment of inpatients with MD and TLE. Seventy-five patients received standard treatment with citalopram, mirtazapine, or reboxetine, respectively, at recommended dosage. Examinations were done with the Hamilton Rating Scale for Depression at admission and after 4 and 20-30 weeks. Plasma levels of anticonvulsants were examined at admission and discharge. Seizures were documented. The antidepressive treatment was efficacious in all antidepressant groups. No case of serious adverse event or drug interaction occurred. There was no increase in frequency or severity of seizures. At endpoint the dropout rate for mirtazapine was significantly higher than that for reboxetine or citalopram. Reboxetine showed a trend to be more efficacious than citalopram but not mirtazapine at Week 4.
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Antidepresivos/uso terapéutico , Trastorno Depresivo Mayor/tratamiento farmacológico , Epilepsia del Lóbulo Temporal/tratamiento farmacológico , Mianserina/análogos & derivados , Adulto , Anciano , Análisis de Varianza , Anticonvulsivantes/sangre , Anticonvulsivantes/uso terapéutico , Antidepresivos/sangre , Distribución de Chi-Cuadrado , Citalopram/uso terapéutico , Demografía , Trastorno Depresivo Mayor/sangre , Manual Diagnóstico y Estadístico de los Trastornos Mentales , Interacciones Farmacológicas , Electroencefalografía , Epilepsia del Lóbulo Temporal/sangre , Femenino , Humanos , Masculino , Mianserina/uso terapéutico , Persona de Mediana Edad , Mirtazapina , Morfolinas/uso terapéutico , Pacientes Desistentes del Tratamiento/estadística & datos numéricos , Inventario de Personalidad , Estudios Prospectivos , Escalas de Valoración Psiquiátrica , Reboxetina , Factores de TiempoRESUMEN
Cocaine metabolism has been studied previously with respect to the formation of predominant hydrolytic or hepatotoxic metabolites via oxidative pathways. In the present study, cocaine and eight of its metabolites (norcocaine, ecgonine methyl ester, benzoylecgonine, benzoylnorecgonine, 3-hydroxy-benzoylecgonine, cocaethylene, norcocaethylene, and ecgonine ethyl ester) were incubated with microsomes from rat liver, kidney, lung, and brain. Qualitative analysis of the metabolites produced was performed using solid phase extraction (SPE), trimethylsilylation, and GC/MS. It was found that the metabolites with a free carboxylic group (e.g., benzoylecgonine) were not further oxidized by microsomal enzymes and their presence in urine or blood may therefore be due to hydrolysis of the respective alkylated entities. Although microsomes from all organs exhibited oxidative metabolism, significant differences were noted. Kidney microsomes produced essentially the same results as liver, but aryl hydroxylated metabolites were not found in incubations with lung and brain microsomes. N-Hydroxy-norcocaine was found only in traces with brain microsomes. It appears that cocaine is converted to N-hydroxy-norcocaine (which is the precursor of toxic metabolites) not only in the liver but also in other organs of rat. This might be relevant in the development of lung toxicity observed in smokers of cocaine ("crack").
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Cocaína/metabolismo , Microsomas/metabolismo , Animales , Encéfalo/metabolismo , Cocaína/análogos & derivados , Cocaína/sangre , Cocaína/orina , Cromatografía de Gases y Espectrometría de Masas , Hidrólisis , Riñón/metabolismo , Pulmón/metabolismo , Masculino , Microsomas/efectos de los fármacos , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
Stimulation of phospholipase C (PLC) by G(q)-coupled receptors such as the M(3) muscarinic acetylcholine receptor (mAChR) is caused by direct activation of PLC-beta enzymes by Galpha(q) proteins. We have recently shown that G(s)-coupled receptors can stimulate PLC-epsilon, apparently via formation of cyclic AMP and activation of the Ras-related GTPase Rap2B. Here we report that PLC stimulation by the M(3) mAChR expressed in HEK-293 cells also involves, in part, similar mechanisms. M(3) mAChR-mediated PLC stimulation and [Ca(2+)](i) increase were reduced by 2',5'-dideoxyadenosine (dd-Ado), a direct adenylyl cyclase inhibitor. On the other hand, overexpression of Galpha(s) or Epac1, a cyclic AMP-regulated guanine nucleotide exchange factor for Rap GTPases, enhanced M(3) mAChR-mediated PLC stimulation. Inactivation of Ras-related GTPases with clostridial toxins suppressed the M(3) mAChR responses. The inhibitory toxin effects were mimicked by expression of inactive Rap2B, but not of other inactive GTPases (Rac1, Ras, RalA, Rap1A, and Rap2A). Activation of the M(3) mAChR induced GTP loading of Rap2B, an effect strongly enhanced by overexpression of Galpha(s) and inhibited by dd-Ado. Overexpression of PLC-epsilon and PLC-beta1, but not PLC-gamma1 or PLC-delta1, enhanced M(3) mAChR-mediated PLC stimulation and [Ca(2+)](i) increase. In contrast, expression of a catalytically inactive PLC-epsilon mutant reduced PLC stimulation by the M(3) mAChR and abrogated the potentiating effect of Galpha(s). In conclusion, our findings suggest that PLC stimulation by the M(3) mAChR is a composite action of PLC-beta1 stimulation by Galpha(q) and stimulation of PLC-epsilon apparently mediated by G(s)-dependent cyclic AMP formation and subsequent activation of Rap2B.