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1.
Am J Respir Crit Care Med ; 187(12): 1324-34, 2013 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-23611140

RESUMEN

RATIONALE: S100A12 is overexpressed during inflammation and is a marker of inflammatory disease. Furthermore, it has been ascribed to the group of damage-associated molecular pattern molecules that promote inflammation. However, the exact role of human S100A12 during early steps of immune activation and sepsis is only partially described thus far. OBJECTIVES: We analyzed the activation of human monocytes by granulocyte-derived S100A12 as a key function of early inflammatory processes and the development of sepsis. METHODS: Circulating S100A12 was determined in patients with sepsis and in healthy subjects with experimental endotoxemia. The release of human S100A12 from granulocytes as well as the promotion of inflammation by activation of human monocytes after specific receptor interaction was investigated by a series of in vitro experiments. MEASUREMENTS AND MAIN RESULTS: S100A12 rises during sepsis, and its expression and release from granulocytes is rapidly induced in vitro and in vivo by inflammatory challenge. A global gene expression analysis of S100A12-activated monocytes revealed that human S100A12 induces inflammatory gene expression. These effects are triggered by an interaction of S100A12 with Toll-like receptor 4 (TLR4). Blocking S100A12 binding to TLR4 on monocytes or TLR4 expressing cell lines (HEK-TCM) abrogates the respective inflammatory signal. On the contrary, blocking S100A12 binding to its second proposed receptor (receptor for advanced glycation end products [RAGE]) has no significant effect on inflammatory signaling in monocytes and RAGE-expressing HEK293 cells. CONCLUSIONS: Human S100A12 is an endogenous TLR4 ligand that induces monocyte activation, thereby acting as an amplifier of innate immunity during early inflammation and the development of sepsis.


Asunto(s)
Inflamación/etiología , Monocitos/fisiología , Proteínas S100/fisiología , Sepsis/inmunología , Receptor Toll-Like 4/fisiología , Adulto , Anciano , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas S100/sangre , Proteína S100A12 , Sepsis/sangre , Receptor Toll-Like 4/sangre , Adulto Joven
2.
Immunology ; 137(2): 172-82, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22804476

RESUMEN

Interactions between danger-associated molecular patterns (DAMP) and pathogen-associated molecular patterns (PAMP) and pattern recognition receptors such as Toll-like receptors (TLRs) are critical for the regulation of the inflammatory process via activation of nuclear factor-κB (NF-κB) and cytokine secretion. In this report, we investigated the capacity of lipopolysaccharide (LPS) -free S100A9 (DAMP) protein to activate human and mouse cells compared with lipoprotein-free LPS (PAMP). First, we showed that LPS and S100A9 were able to increase NF-κB activity followed by increased cytokine and nitric oxide (NO) secretion both in human THP-1 cells and in mouse bone marrow-derived dendritic cells. Surprisingly, although S100A9 triggered a weaker cytokine response than LPS, we found that S100A9 more potently induced IκBα degradation and hence NF-κB activation. Both the S100A9-induced response and the LPS-induced response were completely absent in TLR4 knockout mice, whereas it was only slightly affected in RAGE knockout mice. Also, we showed that LPS and S100A9 NF-κB induction were strongly reduced in the presence of specific inhibitors of TLR-signalling. Chloroquine reduced S100A9 but not LPS signalling, indicating that S100A9 may need to be internalized to be fully active as a TLR4 inducer. This was confirmed using A488-labelled S100A9 that was internalized in THP-1 cells, showing a raise in fluorescence after 30 min at 37°. Chloroquine treatment significantly reduced the fluorescence. In summary, our data indicate that both human and mouse S100A9 are TLR4 agonists. Importantly, S100A9 induced stronger NF-κB activation albeit weaker cytokine secretion than LPS, suggesting that S100A9 and LPS activated NF-κB in a qualitatively distinct manner.


Asunto(s)
Calgranulina B/inmunología , FN-kappa B/inmunología , Receptor Toll-Like 4/inmunología , Animales , Calgranulina B/genética , Calgranulina B/metabolismo , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Humanos , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Transducción de Señal , Receptor Toll-Like 4/deficiencia
3.
PLoS Biol ; 7(4): e97, 2009 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-19402754

RESUMEN

Despite more than 25 years of research, the molecular targets of quinoline-3-carboxamides have been elusive although these compounds are currently in Phase II and III development for treatment of autoimmune/inflammatory diseases in humans. Using photoaffinity cross-linking of a radioactively labelled quinoline-3-carboxamide compound, we could determine a direct association between human S100A9 and quinoline-3-carboxamides. This interaction was strictly dependent on both Zn++ and Ca++. We also show that S100A9 in the presence of Zn++ and Ca++ is an efficient ligand of receptor for advanced glycation end products (RAGE) and also an endogenous Toll ligand in that it shows a highly specific interaction with TLR4/MD2. Both these interactions are inhibited by quinoline-3-carboxamides. A clear structure-activity relationship (SAR) emerged with regard to the binding of quinoline-3-carboxamides to S100A9, as well as these compounds potency to inhibit interactions with RAGE or TLR4/MD2. The same SAR was observed when the compound's ability to inhibit acute experimental autoimmune encephalomyelitis in mice in vivo was analysed. Quinoline-3-carboxamides would also inhibit TNFalpha release in a S100A9-dependent model in vivo, as would antibodies raised against the quinoline-3-carboxamide-binding domain of S100A9. Thus, S100A9 appears to be a focal molecule in the control of autoimmune disease via its interactions with proinflammatory mediators. The specific binding of quinoline-3-carboxamides to S100A9 explains the immunomodulatory activity of this class of compounds and defines S100A9 as a novel target for treatment of human autoimmune diseases.


Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Calgranulina B/metabolismo , Factores Inmunológicos/farmacología , Inflamación/metabolismo , Quinolinas/metabolismo , Receptores Inmunológicos/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Enfermedades Autoinmunes/metabolismo , Calcio/metabolismo , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/efectos adversos , Antígeno 96 de los Linfocitos/metabolismo , Ratones , Ratones Noqueados , Monocitos/metabolismo , Quinolinas/química , Quinolinas/farmacología , Quinolinas/uso terapéutico , Receptor para Productos Finales de Glicación Avanzada , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Zinc/metabolismo
4.
Nutrients ; 13(12)2021 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-34959789

RESUMEN

There is evidence that both omega-3 polyunsaturated fatty acids (n-3 PUFAs) and choline can influence sports performance, but information establishing their combined effects when given in the form of krill oil during power training protocols is missing. The purpose of this study was therefore to characterize n-3 PUFA and choline profiles after a one-hour period of high-intensity physical workout after 12 weeks of supplementation. Thirty-five healthy power training athletes received either 2.5 g/day of Neptune krill oilTM (550 mg EPA/DHA and 150 mg choline) or olive oil (placebo) in a randomized double-blind design. After 12 weeks, only the krill oil group showed a significant HS-Omega-3 Index increase from 4.82 to 6.77% and a reduction in the ARA/EPA ratio (from 50.72 to 13.61%) (p < 0.001). The krill oil group showed significantly higher recovery of choline concentrations relative to the placebo group from the end of the first to the beginning of the second exercise test (p = 0.04) and an 8% decrease in total antioxidant capacity post-exercise versus 21% in the placebo group (p = 0.35). In conclusion, krill oil can be used as a nutritional strategy for increasing the HS-Omega-3 Index, recover choline concentrations and address oxidative stress after intense power trainings.


Asunto(s)
Rendimiento Atlético/fisiología , Colina/administración & dosificación , Euphausiacea , Aceites de Pescado/administración & dosificación , Entrenamiento de Intervalos de Alta Intensidad , Adulto , Animales , Antioxidantes/metabolismo , Colina/sangre , Suplementos Dietéticos , Método Doble Ciego , Ácidos Grasos Omega-3/sangre , Femenino , Voluntarios Sanos , Humanos , Masculino
5.
Langmuir ; 26(5): 3753-9, 2010 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-20175580

RESUMEN

We show how to use well-defined conjugated polyelectrolytes (CPEs) combined with surface energy patterning to fabricate DNA chips utilizing fluorescence signal amplification. Cholesterol-modified DNA strands in complex with a CPE are adsorbed to a surface energy pattern, formed by printing with soft elastomer stamps. Hybridization of the surface bound DNA strands with a short complementary strand from solution is monitored using both fluorescence microscopy and imaging surface plasmon resonance. The CPEs act as antennas, enhancing resonance energy transfer to the dye-labeled DNA when complementary hybridization of the double strand occurs.


Asunto(s)
Dimetilpolisiloxanos/química , Electrólitos/química , Transferencia Resonante de Energía de Fluorescencia , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Acetatos/metabolismo , Adsorción , Secuencia de Bases , Carbocianinas/metabolismo , Colesterol/metabolismo , Cromonas/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Microscopía Fluorescente , Hibridación de Ácido Nucleico , Resonancia por Plasmón de Superficie
6.
Small ; 5(1): 96-103, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19040213

RESUMEN

The organization of conjugated polyelectrolytes (CPEs) interacting with biomolecules sets conditions for the biodetection of biological processes and identity, through the use of optical emission from the CPE. Herein, a well-defined CPE and its binding to DNA is studied. By using dynamic light scattering and circular dichroism spectroscopy, it is shown that the CPE forms a multimolecule ensemble in aqueous solution that is more than doubled in size when interacting with a small DNA chain, while single chains are evident in ethanol. The related changes in the fluorescence spectra upon polymer aggregation are assigned to oscillator strength redistribution between vibronic transitions in weakly coupled H-aggregates. An enhanced single-molecule spectroscopy technique that allows full control of excitation and emission light polarization is applied to combed and decorated lambdaDNA chains. It is found that the organization of combed CPE-lambdaDNA complexes (when dry on the surface) allows considerable variation of CPE distances and direction relative to the DNA chain. By analysis of the polarization data energy transfer between the polymer chains in individual complexes is confirmed and their sizes estimated.


Asunto(s)
ADN/química , Polímeros/química , Tiofenos/química , Dicroismo Circular , Transferencia de Energía , Nanotecnología , Espectrometría de Fluorescencia
7.
Commun Biol ; 2: 176, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31098409

RESUMEN

Innate immune responses are rapid, dynamic and highly regulated to avoid overt reactions. This regulation is executed by innate immune tolerance mechanisms that remain obscure. Wnt5a is a signalling protein mainly involved in developmental processes and cancer. The effect of Wnt5a on inflammatory myeloid cells is controversial. Here, we combine primary cell cultures, in vitro binding studies, mass spectrometry and Drosophila protein modelling to show that Wnt5a is a direct ligand of toll-like receptor (TLR) 2 and 4. The binding promotes a MyD88-non-canonical nuclear factor of kappa B (NFκB) and AP-1 signalling cascade, with contradictory profiles in mouse (pro-inflammatory) and human (anti-inflammatory) myeloid immune cells. These data reveal that the true nature of Wnt5a in inflammatory cells, is to regulate TLR signals, and in human myeloid cells it acts as an endogenous, tolerance-associated molecular pattern (TAMP), inducing IL-10 and innate immune tolerance.


Asunto(s)
Células Mieloides/inmunología , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Proteína Wnt-5a/inmunología , Animales , Células Cultivadas , Citocinas/biosíntesis , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Humanos , Tolerancia Inmunológica , Inmunidad Innata , Interleucina-10/biosíntesis , Interleucina-10/genética , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ligandos , Ratones , Modelos Inmunológicos , Modelos Moleculares , Células Mieloides/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/inmunología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Receptores Toll-Like/química , Receptores Toll-Like/metabolismo , Transcripción Genética , Proteína Wnt-5a/metabolismo
8.
Small ; 3(2): 318-25, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17262758

RESUMEN

The amyloid-like fibril is a biomolecular nanowire template of very high stability. Here we describe the coordination of a conjugated polyelectrolyte, poly(thiophene acetic acid) (PTAA), to bovine insulin fibrils with widths of <10 nm and lengths of up to more than 10 microm. Fibrils complexed with PTAA are aligned on surfaces through molecular combing and transfer printing. Single-molecule spectroscopy techniques are applied to chart spectral variation in the emission of these wires. When these results are combined with analysis of the polarization of the emitted light, we can conclude that the polymer chains are preferentially aligned along the fibrillar axis.


Asunto(s)
Acetatos/química , Péptidos beta-Amiloides/química , Materiales Biomiméticos/química , Cristalización/métodos , Nanoestructuras/química , Nanoestructuras/ultraestructura , Nanotecnología/métodos , Tiofenos/química , Animales , Bovinos , Sustancias Macromoleculares/química , Ensayo de Materiales , Conformación Molecular , Tamaño de la Partícula , Polímeros/química , Propiedades de Superficie
9.
J Biomol Screen ; 12(4): 464-72, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-17435172

RESUMEN

Protein-protein interactions are widely found in biological systems controlling diverse cellular events. Because these interactions are implicated in many diseases such as autoimmunity and cancer, regulation of protein-protein interactions provides ideal targets for drug intervention. The CD80-CD28 costimulatory pathway plays a critical role in regulation of the immune response and thus constitutes an attractive target for therapeutic manipulation of autoimmune diseases. The objective of this study is to identify small compounds disrupting these pivotal protein-protein interactions. Compounds that specifically blocked binding of CD80 to CD28 were identified using a strategy involving a cell-based scintillation proximity assay as the initial step. Secondary screening (e.g., by analyzing the direct binding of these compounds to the target immobilized on a biosensor surface) revealed that these compounds are highly selective CD80 binders. Screening of structurally related derivatives led to the identification of the chemical features required for inhibition of the CD80-CD28 interaction. In addition, the optimization process led to a 10-fold increase in binding affinity of the CD80 inhibitors. Using this approach, the authors identify low-molecular-weight compounds that specifically and with high potency inhibit the interaction between CD80 and CD28. These compounds serve as promising starting points for further development of CD80 inhibitors as potential immunomodulatory drugs.


Asunto(s)
Antígeno B7-1/metabolismo , Diseño de Fármacos , Animales , Unión Competitiva , Células CHO , Cricetinae , Cricetulus , Cabras , Humanos , Ratones , Unión Proteica , Mapeo de Interacción de Proteínas , Conejos
10.
Small ; 2(8-9): 1068-74, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17193170

RESUMEN

Aligned and stretched lambda DNA is directed to specific locations on solid substrates. Surface-energy modification of glass substrates by using patterned polydimethylsiloxane (PDMS) stamps is used to direct DNA onto the surface-energy-modified micrometer-scale pattern through molecular combing. As an alternative, patterned and nonpatterned PDMS stamps modified with polymethylmethacrylate (PMMA) are utilized to direct the stretched DNA to the desired location and the results are compared. The DNA is elongated through molecular combing on the stamp and transfer printed onto the surfaces. PMMA-modified stamps show a more defined length of the stretched DNA, as compared to bare PDMS stamps. A combination of these two methods is also demonstrated. As an application example, transfer printing of DNA decorated with a semiconducting conjugated polyelectrolyte is shown. The resulting patterned localization of stretched DNA can be utilized for functional nanodevice structures, as well as for biological applications.


Asunto(s)
ADN , Impresión , Dimetilpolisiloxanos , Metabolismo Energético , Polimetil Metacrilato , Siliconas , Propiedades de Superficie
11.
Biosens Bioelectron ; 82: 55-63, 2016 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-27040942

RESUMEN

We present a simple and inexpensive method for label-free detection of biomolecules. The method monitors the changes in streaming current in a fused silica capillary as target biomolecules bind to immobilized receptors on the inner surface of the capillary. To validate the concept, we show detection and time response of different protein-ligand and protein-protein systems: biotin-avidin and biotin-streptavidin, barstar-dibarnase and Z domain-immunoglobulin G (IgG). We show that specific binding of these biomolecules can be reliably monitored using a very simple setup. Using sequential injections of various proteins at a diverse concentration range and as well as diluted human serum we further investigate the capacity of the proposed technique to perform specific target detection from a complex sample. We also investigate the time for the signal to reach equilibrium and its dependence on analyte concentration and demonstrate that the current setup can be used to detect biomolecules at a concentration as low as 100pM without requiring any advanced device fabrication procedures. Finally, an analytical model based on diffusion theory has been presented to explain the dependence of the saturation time on the analyte concentration and capillary dimensions and how reducing length and inner diameter of the capillary is predicted to give faster detection and in practice also lower limit of detection.


Asunto(s)
Técnicas Biosensibles/instrumentación , Proteínas/análisis , Avidina/análisis , Bacillus amyloliquefaciens/enzimología , Proteínas Bacterianas/análisis , Biotina/análisis , Diseño de Equipo , Humanos , Inmunoglobulina G/análisis , Ligandos , Ribonucleasas/análisis , Proteína Estafilocócica A/análisis , Staphylococcus aureus/química , Estreptavidina/análisis , Streptomyces/química
12.
PLoS One ; 11(5): e0156377, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27228163

RESUMEN

The cytosolic Ca2+-binding S100A9 and S100A8 proteins form heterodimers that are primarily expressed in human neutrophils and monocytes. We have recently shown that S100A9 binds to TLR4 in vitro and induces TLR4-dependent NF-κB activation and a pro-inflammatory cytokine response in monocytes. In the present report we have further investigated the S100A9-mediated stimulation of TLR4 in monocytes. Using transmission immunoelectron microscopy, we detected focal binding of S100A9 to monocyte membrane subdomains containing the caveolin-1 protein and TLR4. Furthermore, the S100A9 protein was detected in early endosomes of the stimulated cells, indicating that the protein could be internalized by endocytosis. Although stimulation of monocytes with S100A9 was strictly TLR4-dependent, binding of S100A9 to the plasma membrane and endocytosis of S100A9 was still detectable and coincided with CD14 expression in TLR4-deficient cells. We therefore investigated whether CD14 would be involved in the TLR4-dependent stimulation and could show that the S100A9-induced cytokine response was inhibited both in CD14-deficient cells and in cells exposed to CD14 blocking antibodies. Further, S100A9 was not internalized into CD14-deficient cells suggesting a direct role of CD14 in endocytosis of S100A9. Finally, we could detect satiable binding of S100A9 to CD14 in surface plasmon resonance experiments. Taken together, these results indicate that CD14 is a co-receptor of TLR4 in the S100A9-induced cytokine response.


Asunto(s)
Calgranulina B/inmunología , Receptores de Lipopolisacáridos/inmunología , Monocitos/inmunología , Receptor Toll-Like 4/inmunología , Animales , Calgranulina B/genética , Caveolina 1/genética , Caveolina 1/inmunología , Línea Celular Tumoral , Membrana Celular/genética , Membrana Celular/inmunología , Citocinas/genética , Citocinas/inmunología , Humanos , Inflamación/genética , Inflamación/inmunología , Receptores de Lipopolisacáridos/genética , Ratones , Ratones Noqueados , Unión Proteica , Resonancia por Plasmón de Superficie , Receptor Toll-Like 4/genética
13.
Micromachines (Basel) ; 7(8)2016 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-30404306

RESUMEN

We present a novel microfluidic system that integrates droplet microfluidics with a silicon nanoribbon field-effect transistor (SiNR FET), and utilize this integrated system to sense differences in pH. The device allows for selective droplet transfer to a continuous water phase, actuated by dielectrophoresis, and subsequent detection of the pH level in the retrieved droplets by SiNR FETs on an electrical sensor chip. The integrated microfluidic system demonstrates a label-free detection method for droplet microfluidics, presenting an alternative to optical fluorescence detection. In this work, we were able to differentiate between droplet trains of one pH-unit difference. The pH-based detection method in our integrated system has the potential to be utilized in the detection of biochemical reactions that induce a pH-shift in the droplets.

14.
Biosens Bioelectron ; 20(9): 1764-71, 2005 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-15681192

RESUMEN

A water-soluble polythiophene, POWT, with zwitterionic peptide like side chains possess good characteristics for biosensor applications. The zwitterionic side chains of the polymer can couple to biomolecules via electrostatic and hydrogen bonding. This creates possibilities to imprint biomolecules to spin-coated polymer films with maintained functionality, and use the resulting matrix as a biosensor. Polymer-biomolecular interaction studies done with surface plasmon resonance (SPR) reveal a well performing sensor matrix with high affinity for DNA hybridizations as well as for protein detection. The responses are distinct and very specific. A directional dependence of antibodies binding to POWT layer has also been observed. The polymer films have also been characterized by optical methods. Emission and absorption measurements in different buffer systems confirm that the polymer matrix can undergo structural and conformational changes on surfaces. The dielectric function in the interval 300-800 nm of POWT is reported, based on variable angle spectroscopic ellipsometry. This modeling reveals that a considerable amount of water is included in the material. The polymer layer possesses the characteristics needed for biochip applications and micro array techniques.


Asunto(s)
Técnicas Biosensibles/métodos , ADN/análisis , Inmunoensayo/métodos , Mediciones Luminiscentes/métodos , Polímeros/química , Resonancia por Plasmón de Superficie/métodos , Tiofenos/química , Materiales Biocompatibles/química , Técnicas Biosensibles/instrumentación , Electrólitos/química , Inmunoensayo/instrumentación , Mediciones Luminiscentes/instrumentación , Ensayo de Materiales , Resonancia por Plasmón de Superficie/instrumentación , Propiedades de Superficie
15.
PLoS One ; 10(12): e0145217, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26661255

RESUMEN

We show here, by using surface biotinylation, followed by Western blotting or surface plasmon resonance analysis, that very low levels of S100A8 and/or S100A9 can be detected on the surface of THP-1 cells or freshly isolated human monocytes. This was supported by immune-electron microscopy where we observed membrane-associated expression of the proteins restricted to small patches. By using confocal microscopy we could determine that S100A8 and S100A9 protein in THP-1 cells or freshly isolated human monocytes was mostly present in vesicular structures. This finding was confirmed using immune-electron microscopy. Subcellular fractionation and confocal microscopy showed that these vesicular structures are mainly early endosomes and endolysosomes. Our subsequent studies showed that accumulation of S100A8 and S100A9 in the endolysosomal compartment is associated with induction of their release from the cells. Furthermore, an inhibitor of lysosomal activity could modulate the release of S100A8 and S100A9 in the extracellular milieu. Our current results suggest that the S100A8 and S100A9 proteins are primarily associated with certain kinds of cytosolic vesicles and may be secreted via an endolysosomal pathway.


Asunto(s)
Calgranulina A/metabolismo , Calgranulina B/metabolismo , Transporte Biológico , Biotinilación , Western Blotting , Calgranulina A/química , Calgranulina A/genética , Calgranulina B/química , Calgranulina B/genética , Células Cultivadas , Dimerización , Ensayo de Inmunoadsorción Enzimática , Humanos , Interleucina-10/farmacología , Lisosomas/metabolismo , Microscopía Confocal , Microscopía Electrónica , Monocitos/citología , Monocitos/metabolismo , Resonancia por Plasmón de Superficie , Factor de Necrosis Tumoral alfa/farmacología , Ultracentrifugación , Regulación hacia Arriba/efectos de los fármacos
16.
J Neurotrauma ; 30(5): 392-402, 2013 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-23057993

RESUMEN

Abstract The molecular processes involved in axonal regeneration after traumatic brain injury (TBI) are still not fully understood. In this study, we have established a novel in vitro injury model of TBI based on microcontact printing (µCP) that enables close-up investigations of injured neurons. The model is also suitable for quantitative measurements of axonal outgrowth, making it a useful tool in the studies of basic mechanisms behind axonal regeneration. Cortical neurons from mouse embryos are cultured on µCP cover-slips for 8 days, and the neurons are then injured in a precise manner using a thin plastic tip that does not affect the µCP pattern of extracellular matrix proteins. By close-up time-lapse experiments and immunostainings, we show that the neurons have a tremendous capacity to regenerate their neurites after injury. The cut induces growth cone formation, and the regenerating axons strictly follow the µCP pattern. Moreover, by using the injury model, we demonstrate that hydrogen peroxide (H2O2) decreases axonal regeneration after injury without affecting the neurons' ability to form growth cones. Co-culture with glial cells does not rescue the axonal regeneration, indicating that the mechanism by which H2O2 affects axonal regeneration differ from its cytotoxic effect.


Asunto(s)
Axones/metabolismo , Modelos Animales de Enfermedad , Peróxido de Hidrógeno/metabolismo , Regeneración Nerviosa/fisiología , Animales , Axones/efectos de los fármacos , Lesiones Encefálicas , Células Cultivadas , Técnicas de Cocultivo , Peróxido de Hidrógeno/farmacología , Inmunohistoquímica , Ratones , Microscopía Fluorescente , Regeneración Nerviosa/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo
17.
Atherosclerosis ; 228(1): 69-79, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23497784

RESUMEN

OBJECTIVE: There is an emerging widespread interest in the role of damage-associated molecular pattern molecules (DAMP) S100A8, S100A9 and S100A12 in cardiovascular and other diseases. In this study we tested the efficacy of ABR-215757, a S100 protein binding immuno-modulatory compound to stabilize atherosclerosis in transgenic ApoE null mice that express the human pro-inflammatory S100A12 protein within the smooth muscle cell (SM22α-S100A12). METHODS: Twelve-week old S100A12 transgenic/ApoE(-/-) and WT/ApoE(-/-) mice were treated with ABR-21575 for 5 weeks and were analyzed 4 month later. RESULTS: Surface plasmon resonance analysis demonstrated that S100A12 interacts with ABR-215757 in a zinc dependent manner in vitro. In vivo, ABR-215757 administration reduced features of advanced plaque morphology resulting in smaller necrotic cores, diminished intimal and medial vascular calcification, and reduced amount of infiltrating inflammatory cells. ABR-215757 normalized aortic expression of RAGE protein and normalized experimentally-induced delayed hypersensitivity. The effect of ABR-215757 was more prominent in ApoE(-/-) mice expressing S100A12 than in ApoE(-/-) animals lacking expression of human S100A12 protein. CONCLUSION: Our data suggest that S100A12 is important for progression of atherosclerosis and can be targeted by the small molecule ABR-215757. The specific binding of quinoline-3-carboxamides to S100A12 attenuates S100A12-mediated features of accelerated murine atherosclerosis.


Asunto(s)
Apolipoproteínas E/genética , Aterosclerosis/tratamiento farmacológico , Placa Aterosclerótica/tratamiento farmacológico , Quinolinas/farmacología , Proteínas S100/genética , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/patología , Aorta Torácica/fisiología , Aterosclerosis/genética , Aterosclerosis/patología , Calgranulina B/metabolismo , Modelos Animales de Enfermedad , Humanos , Inmunosupresores/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Placa Aterosclerótica/genética , Placa Aterosclerótica/patología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/genética , Proteínas Recombinantes/genética , Proteínas S100/metabolismo , Proteína S100A12 , Vasculitis/tratamiento farmacológico , Vasculitis/genética , Vasculitis/patología , Zinc/metabolismo
18.
PLoS One ; 8(5): e63012, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23667563

RESUMEN

S100A4 and S100A9 proteins have been described as playing roles in the control of tumor growth and metastasis. We show here that a chemical probe, oxyclozanide (OX), selected for inhibiting the interaction between S100A9 and the receptor for advanced glycation end-products (RAGE) interacts with both S100A9 and S100A4. Furthermore, we show that S100A9 and S100A4 interact with RAGE and TLR4; interactions that can be inhibited by OX. Hence, S100A4 and S100A9 display similar functional elements despite their primary sequence diversity. This was further confirmed by showing that S100A4 and S100A9 dimerize both in vitro and in vivo. All of these interactions required levels of Zn++ that are found in the extracellular space but not intracellularly. Interestingly, S100A4 and S100A9 are expressed by distinct CD11b+ subpopulations both in healthy animals and in animals with either inflammatory disease or tumor burden. The functions of S100A9 and S100A4 described in this paper, including heterodimerization, may therefore reflect S100A9 and S100A4 that are released into the extra-cellular milieu.


Asunto(s)
Calgranulina B/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Linfoma/metabolismo , Sondas Moleculares/metabolismo , Oxiclozanida/metabolismo , Proteínas S100/metabolismo , Animales , Western Blotting , Antígeno CD11b/metabolismo , Calgranulina B/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dimerización , Líquido Extracelular/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Oxiclozanida/farmacología , Proteína de Unión al Calcio S100A4 , Proteínas S100/química , Receptor Toll-Like 4/metabolismo , Zinc/metabolismo
19.
PLoS One ; 8(10): e79082, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24194959

RESUMEN

The T lymphocytes are the most important effector cells in immunotherapy of cancer. The conceptual objective for developing the tumor targeted superantigen (TTS) ABR-217620 (naptumomab estafenatox, 5T4Fab-SEA/E-120), now in phase 3 studies for advanced renal cell cancer, was to selectively coat tumor cells with cytotoxic T lymphocytes (CTL) target structures functionally similar to natural CTL pMHC target molecules. Here we present data showing that the molecular basis for the anti-tumor activity by ABR-217620 resides in the distinct interaction between the T cell receptor ß variable (TRBV) 7-9 and the engineered superantigen (Sag) SEA/E-120 in the fusion protein bound to the 5T4 antigen on tumor cells. Multimeric but not monomeric ABR-217620 selectively stains TRBV7-9 expressing T lymphocytes from human peripheral blood similar to antigen specific staining of T cells with pMHC tetramers. SEA/E-120 selectively activates TRBV7-9 expressing T lymphocytes resulting in expansion of the subset. ABR-217620 selectively triggers TRBV7-9 expressing cytotoxic T lymphocytes to kill 5T4 positive tumor cells. Furthermore, ABR-217620 activates TRBV7-9 expressing T cell line cells in the presence of cell- and bead-bound 5T4 tumor antigen. Surface plasmon resonance analysis revealed that ABR-217620 binds to 5T4 with high affinity, to TRBV7-9 with low affinity and to MHC class II with very low affinity. The T lymphocyte engagement by ABR-217620 is constituted by displaying high affinity binding to the tumor cells (KD approximately 1 nM) and with the mimicry of natural productive immune TCR-pMHC contact using affinities of around 1 µM. This difference in kinetics between the two components of the ABR-217620 fusion protein will bias the binding towards the 5T4 target antigen, efficiently activating T-cells via SEA/E-120 only when presented by the tumor cells.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Antineoplásicos/inmunología , Citotoxicidad Inmunológica/inmunología , Enterotoxinas/inmunología , Inmunoconjugados/inmunología , Imitación Molecular/inmunología , Neoplasias/inmunología , Superantígenos/inmunología , Linfocitos T/inmunología , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Clonación Molecular , Cartilla de ADN/genética , Enterotoxinas/metabolismo , Citometría de Flujo , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T/genética , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T/inmunología , Humanos , Inmunoconjugados/metabolismo , Cinética , Luciferasas , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Superantígenos/metabolismo , Resonancia por Plasmón de Superficie
20.
Cancer Res ; 73(4): 1386-99, 2013 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-23149916

RESUMEN

Tasquinimod is an orally active antiangiogenic drug that is currently in phase III clinical trials for the treatment of castration-resistant prostate cancer. However, the target of this drug has remained unclear. In this study, we applied diverse strategies to identify the histone deacetylase HDAC4 as a target for the antiangiogenic activity of tasquinimod. Our comprehensive analysis revealed allosteric binding (Kd 10-30 nmol/L) to the regulatory Zn(2+) binding domain of HDAC4 that locks the protein in a conformation preventing HDAC4/N-CoR/HDAC3 complex formation. This binding inhibited colocalization of N-CoR/HDAC3, thereby inhibiting deacetylation of histones and HDAC4 client transcription factors, such as HIF-1α, which are bound at promoter/enhancers where epigenetic reprogramming is required for cancer cell survival and angiogenic response. Through this mechanism, tasquinimod is effective as a monotherapeutic agent against human prostate, breast, bladder, and colon tumor xenografts, where its efficacy could be further enhanced in combination with a targeted thapsigargin prodrug (G202) that selectively kills tumor endothelial cells. Together, our findings define a mechanism of action of tasquinimod and offer a perspective on how its clinical activity might be leveraged in combination with other drugs that target the tumor microenvironment. Cancer Res; 73(4); 1386-99. ©2012 AACR.


Asunto(s)
Histona Desacetilasas/metabolismo , Neoplasias/tratamiento farmacológico , Quinolinas/farmacología , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Acetilación/efectos de los fármacos , Regulación Alostérica , Animales , Western Blotting , Hipoxia de la Célula , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Histona Desacetilasas/química , Histona Desacetilasas/genética , Histonas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Desnudos , Modelos Moleculares , Neoplasias/genética , Neoplasias/patología , Profármacos/farmacología , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Quinolonas , Interferencia de ARN , Proteínas Represoras/química , Proteínas Represoras/genética , Tapsigargina/farmacología , Microambiente Tumoral/genética
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