RESUMEN
BACKGROUND/AIMS: Angiotensin II receptor blockers (ARBs) have been proved to be effective in preventing atrial structural and electrical remodelinq in atrial fibrillation (AF). Previous studies have shown that parasympathetic remodeling plays an important role in AF. However, the effects of ARBs on atrial parasympathetic remodeling in AF and the underlying mechanisms are still unknown. METHODS: Canines were divided into sham-operated, pacing and valsartan + pacing groups. Rats and HL-1 cardiomyocytes were divided into control, angiotensin II (Ang II) and Ang II + valsartan groups, respectively. Atrial parasympathetic remodeling was quantified by immunocytochemical staining with anti-choline acetyltransferase (ChAT) antibody. Western blot was used to analysis the protein expression of neurturin. RESULTS: Both inducibility and duration were increased in chronic atrial rapid-pacing canine model, which was significantly inhibited by the treatment with valsartan. The density of ChAT-positive nerves and the protein level of neurturin in the atria of pacing canines were both increased than those in sham-operated canines. Ang II treatment not only induced atrial parasympathetic remodeling in rats, but also up-regulated the protein expression of neurturin. Valsartan significantly prevented atrial parasympathetic remodeling, and suppressed the protein expression of neurturin. Meanwhile, valsartan inhibited Ang II -induced up-regulation of neurturin and MAPKs in cultured cardiac myocytes. Inhibition of MAPKs dramatically attenuated neurturin up-regulation induced by Ang II. CONCLUSION: Parasympathetic remodeling was present in animals subjected to rapid pacing or Ang II infusion, which was mediated by MAPKs/neurturin pathway. Valsartan is able to prevent atrial parasympathetic remodeling and the occurrence of AF via inhibiting MAPKs/neurturin pathway.
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Fibrilación Atrial/prevención & control , Sistema de Señalización de MAP Quinasas , Neurturina/metabolismo , Valsartán/farmacología , Animales , Perros , Femenino , Masculino , RatasAsunto(s)
Remodelación Atrial/fisiología , Atrios Cardíacos/fisiopatología , Hipertiroidismo/fisiopatología , Hipotiroidismo/fisiopatología , Sistema Nervioso Simpático/fisiopatología , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Atrios Cardíacos/inervación , Hipertiroidismo/complicaciones , Hipertiroidismo/metabolismo , Hipotiroidismo/complicaciones , Hipotiroidismo/metabolismo , Masculino , Ratas , Ratas Wistar , Tirotropina/sangreRESUMEN
OBJECTIVE: To investigate whether microRNAs (miRs) can serve as novel biomarkers for in-stent restenosis (ISR). METHODS: This retrospective, observational single-centre study was conducted at the cardiovascular department of a tertiary hospital centre in the north of China. Follow-up coronary angiography at 6 to 12 months was performed in 181 consecutive patients implanted with drug-eluting stents. Fifty-two healthy volunteers served as the control group. The plasma miRs levels were analyzed by quantitative real-time PCR. Receiver-operating characteristic curve (ROC) analysis was performed to investigate the characters of these miRs as potential biomarkers of ISR. RESULTS: MiR-21 levels in ISR patients were significantly higher than those in non-ISR patients and healthy controls (P<0.05), while miR-100 (P<0.05), miR-143 (P<0.001) and miR-145 (P<0.0001) levels were significantly decreased in ISR patients. Further analysis showed that miR-21 levels were remarkably increased (P = 0.045), while miR-100 (P = 0.041), miR-143 (P = 0.029) and miR-145 (P<0.01) levels were dramatically decreased in patients with diffuse ISR compared to those with focal ISR. ROC analysis demonstrated that the area under curve of miR-145, miR-143, miR-100 and miR-21 were 0.880 (95% confidence interval; CI = 0.791-0.987, P<0.001), 0.818 (95% confidence interval; CI = 0.755-0.963, P<0.001), 0.608 (95% confidence interval; CI = 0.372-0.757, P<0.05) and 0.568 (95% confidence interval; CI = 0.372-0.757, P<0.05), with specificity of 83.1%, 80.1%, 68.9% and 68.6%, and sensitivity of 88.7%, 82.1%, 60.2% and 50.1%, respectively. CONCLUSIONS: Circulating miR-143 and miR-145 levels are associated with the occurrence of ISR and can serve as novel noninvasive biomarkers for ISR.
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Angioplastia Coronaria con Balón , Reestenosis Coronaria/sangre , Stents Liberadores de Fármacos , MicroARNs/sangre , Anciano , Área Bajo la Curva , Biomarcadores/sangre , Estudios de Casos y Controles , Angiografía Coronaria , Reestenosis Coronaria/diagnóstico , Reestenosis Coronaria/diagnóstico por imagen , Femenino , Humanos , Masculino , Persona de Mediana Edad , Curva ROC , Estudios RetrospectivosRESUMEN
The investigation of agents for the treatment of osteoporosis has been a long-standing effort. The Wnt pathway plays an important role in bone formation and regeneration, and expression of Wnt pathway inhibitors, Dickkopf-1 (DKK1), appears to be associated with changes in bone mass. Inactivation of DKK1 leads to substantially increased bone mass in genetically manipulated animals. DKK1-derived peptides (DDPs) were added to BMP2-stimulated MC3T3-E1 preosteoblastic cells in vitro to evaluate inhibitory activity of DDPs in MC3T3-E1 cell differentiation. Study was extended in vivo on old female mice to show whether or not inhibition of endogenous DKK1 biological activity using DDPs vaccination approach leads to increase of bone formation, bone density, and improvement of bone microstructure. We reported that synthetic DDPs were able to reduce alkaline phosphatase activity, prevent mineralization and inhibit the differentiation of MC3T3-E1 cells in vitro. Furthermore, vaccination with these DDPs in aged female mice 4 times for a total period of 22 weeks promoted bone mass and bone microstructure. 3D microCT and histomorphometric analysis showed that there were significant increase in bone mineral densities, improvement of bone microstructure and promotion of bone formation in the vaccinated mice, especially in the mice vaccinated with DDP-A and DDP-C. Histological and scanning electron microscopy image analysis also indicated that vaccination increased trabecular bone mass and significantly decreased fragmentation of bone fibers. Taken together, these preclinical results suggest that vaccination with DDPs represents a promising new therapeutic approach for the treatment of bone-related disorders, such as osteoporosis.
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Péptidos y Proteínas de Señalización Intercelular/inmunología , Osteogénesis/fisiología , Osteoporosis/prevención & control , Vacunas/farmacología , Absorciometría de Fotón , Envejecimiento , Animales , Western Blotting , Modelos Animales de Enfermedad , Femenino , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Rastreo , Osteoporosis/metabolismo , Péptidos/inmunología , Vacunación , Microtomografía por Rayos XRESUMEN
Tyro3, a member of the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases, has emerged as a potential oncogene in melanoma. Here, we confirm that Tyro3 is specifically overexpressed in primary melanoma samples and show that Tyro3 is expressed at varying levels in numerous melanoma cell lines. Short hairpin RNA-mediated knockdown of Tyro3 led to significant cell death via apoptotic mechanisms in nearly all melanoma cell lines tested, regardless of the BRAF or NRAS mutation status or co-expression of Axl and/or Mer. We generated soluble and monomeric versions of the human Tyro3 extracellular domain and human Gas6 for affinity measurements and correlated these values with the level of Gas6 required to induce Tyro3 signaling in cellular assays. Calcium was critical for the correct folding of Gas6 and its binding to Tyro3. In melanoma cell lines, Gas6 induced Tyro3 phosphorylation and downstream Akt phosphorylation without apparent effects on Erk. We generated monoclonal antibodies (mAbs) against Tyro3 to examine their effect on survival signaling in melanoma cell lines. The mAbs generated against Tyro3 included nonligand blockers, partial blockers, and competitive ligand blockers. A number of weak and partial ligand blockers (all recognizing the Tyro3 Ig domains) were the most effective at blocking ligand-mediated downstream signaling of Tyro3. Overall, these data indicate that Tyro3 may confer increased survival signals in melanoma cells and can be stymied using inhibitory mAbs. These mAbs may be useful for further investigations of the role of Tyro3 in melanoma.
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Anticuerpos Monoclonales/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Melanoma/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Western Blotting , Rastreo Diferencial de Calorimetría , División Celular , Línea Celular Tumoral , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoprecipitación , Melanoma/inmunología , Melanoma/patología , Fosforilación , ARN Mensajero/análisis , Proteínas Tirosina Quinasas Receptoras/inmunologíaRESUMEN
NUCB2¹â»8³ has been recently reported as an anorexigenic and anti-hyperglycemic peptide. Here we report that NUCB2¹â»8³ promotes osteogenesis. It was found after two months of once-a-day intravenous injection of NUCB2¹â»8³, bone mineral density of femora and lumbar vertebrae were increased in ovariectomized rats. NUCB2¹â»8³ also increased the alkaline phosphatase activity and promoted mineralization in mouse MC3T3-E1 preosteoblastic cell line. When either both Arg6° and Arg6³ or Ser7² were mutated to Ala, the pro-osteogenic activity was completely lost, indicating that these residues are structurally important for its biological function. Furthermore, it encumbered osteoclastic differentiation of RAW 264.7 macrophage. It also excluded any possibility of the effect caused by contaminants or experimental faults, and demonstrated that the pro-osteogenic activity observed was a specific effect of NUCB2¹â»8³ itself. These findings warranted that further studies on NUCB2¹â»8³ would be valuable for the treatment of bone metabolic diseases especially for osteoporosis.
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Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/farmacología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/farmacología , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/farmacología , Osteogénesis/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Densidad Ósea/efectos de los fármacos , Calcificación Fisiológica/efectos de los fármacos , Proteínas de Unión al Calcio/genética , Línea Celular , Proteínas de Unión al ADN/genética , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Proteínas del Tejido Nervioso/genética , Nucleobindinas , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Ovariectomía , Ligando RANK/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Human studies using Abs to two different, nonoverlapping epitopes of IL-13 suggested that epitope specificity can have a clinically significant impact on clearance of IL-13. We propose that Ab modulation of IL-13 interaction with IL-13Rα2 underlies this effect. Two Abs were administered to healthy subjects and mild asthmatics in separate dose-ranging studies and allergen-challenge studies. IMA-638 allows IL-13 interaction with IL-13Rα1 or IL-13Rα2 but blocks recruitment of IL-4Rα to the IL-13/IL-13Rα1 complex, whereas IMA-026 competes with IL-13 interaction with IL-13Rα1 and IL-13Rα2. We found â¼10-fold higher circulating titer of captured IL-13 in subjects treated with IMA-026 compared with those administered IMA-638. To understand how this difference could be related to epitope, we asked whether either Ab affects IL-13 internalization through cell surface IL-13Rα2. Humans inducibly express cell surface IL-13Rα2 but lack the soluble form that regulates IL-13 responses in mice. Cells with high IL-13Rα2 expression rapidly and efficiently depleted extracellular IL-13, and this activity persisted in the presence of IMA-638 but not IMA-026. The potency and efficiency of this clearance pathway suggest that cell surface IL-13Rα2 acts as a scavenger for IL-13. These findings could have important implications for the design and characterization of IL-13 antagonists.
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Subunidad alfa2 del Receptor de Interleucina-13/metabolismo , Interleucina-13/inmunología , Interleucina-13/metabolismo , Isoanticuerpos/fisiología , Receptores Depuradores/metabolismo , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Relación Dosis-Respuesta Inmunológica , Sistemas de Liberación de Medicamentos , Espacio Extracelular/inmunología , Espacio Extracelular/metabolismo , Células HT29 , Humanos , Interleucina-13/antagonistas & inhibidores , Subunidad alfa2 del Receptor de Interleucina-13/antagonistas & inhibidores , Subunidad alfa2 del Receptor de Interleucina-13/biosíntesis , Macaca fascicularis , Ratones , Ratones Endogámicos BALB C , Receptores Depuradores/antagonistas & inhibidores , Receptores Depuradores/fisiologíaRESUMEN
The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor implicated in a number of diseases including autoimmune diseases. To further understand the pathogenic mechanism of RAGE in these diseases, we searched for additional ligands. We discovered that C3a bound to RAGE with an EC(50) of 1.9 nM in an ELISA, and the binding was increased both in magnitude (by >2-fold) and in affinity (EC(50) 70 pM) in the presence of human stimulatory unmethylated cytosine-guanine-rich DNA A (hCpGAs). Surface plasmon resonance and fluorescence anisotropy analyses demonstrated that hCpGAs could bind directly to RAGE and C3a and form a ternary complex. In human PBMCs, C3a increased IFN-α production in response to low levels of hCpGAs, and this synergy was blocked by soluble RAGE or by an Ab directed against RAGE. IFN-α production was reduced in response to mouse CpGAs and C3a in RAGE(-/-) mouse bone marrow cells compared wild-type mice. Taken together, these data demonstrate that RAGE is a receptor for C3a and CpGA. Through direct interaction, C3a and CpGA synergize to increase IFN-α production in a RAGE-dependent manner and stimulate an innate immune response. These findings indicate a potential role of RAGE in autoimmune diseases that show accumulation of immunostimulatory DNA and C3a.
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Complemento C3a/metabolismo , ADN/metabolismo , Interferón gamma/metabolismo , Oligonucleótidos/metabolismo , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Animales , Complemento C3a/inmunología , ADN/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Interferón gamma/inmunología , Ratones , Ratones Noqueados , Oligonucleótidos/inmunología , Unión Proteica , Receptor para Productos Finales de Glicación Avanzada/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
The peptide apelin is expressed in the pulmonary vasculature and is involved in the pathogenesis of many cardiovascular diseases. It has a biphasic role in the regulation of vasomotor tone related to the vascular endothelium. In this study, we induced acute pulmonary embolism (APE) in dogs with autologous blood clots to assess the effect of apelin on pulmonary and systemic circulation in the acute phase of APE. The expression of apelin mRNA was found to be upregulated in the lung tissue in the early several hours after APE induction and decreased at 24 h. The expression of apelin protein in the pulmonary arteries did not change within 24 h after APE, but significantly increased in the bronchial epithelial cells as early as 1h and decreased at 24 h. In normal anesthetized dogs, intravenous bolus administration of apelin significantly reduced the mean arterial pressure (MAP), but did not significantly affect the mean pulmonary arterial pressure (MPAP). In the dogs with APE, apelin decreased MPAP, whereas its impact on MAP was not significantly different from that in the control group. Taken together, the level of endogenous apelin did not change significantly in the pulmonary arterial wall, whereas its expression in the bronchial epithelium was upregulated in the early stage of APE. The effect of exogenous apelin on vasomotor tone was complicated: it resulted in differential changes in the pulmonary and systemic arterial pressures under different physiological and pathological conditions.
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Hemodinámica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/fisiología , Pulmón/metabolismo , Péptidos/fisiología , Circulación Pulmonar/efectos de los fármacos , Embolia Pulmonar/fisiopatología , Receptores Acoplados a Proteínas G/fisiología , Animales , Bronquios/irrigación sanguínea , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Bronquios/patología , Perros , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Epitelio/patología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/administración & dosificación , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/farmacología , Ligandos , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Pulmón/patología , Masculino , Especificidad de Órganos , Péptidos/genética , Embolia Pulmonar/tratamiento farmacológico , Embolia Pulmonar/metabolismo , Embolia Pulmonar/patología , ARN Mensajero/metabolismo , Distribución Aleatoria , Receptores Acoplados a Proteínas G/genética , Factores de TiempoRESUMEN
PURPOSE: This study was designed to evaluate the effects of a calpain inhibitor on cardiac muscle apoptosis in rapid pacing canine atrial fibrillation (AF) models. METHODS: Twenty one dogs were divided into three groups: a sham operation group, a control AF group and a calpain inhibitor group. Sustained AF was induced by rapid right atrium pacing at 600 beats per minute. N-Acetyl-Leu-Leu-Met (1.0 mg/kg/day) was administered in the calpain inhibitor group for three weeks. The activity of calpain I and cardiomyocyte apoptosis were measured by fluorometry and TUNEL assay, respectively. Protein expression of caspase-3 was detected by Western blot. The localizations of caspase-3, caspase-8, bcl-2 and ARC were assessed by immunohistochemistry. RESULTS: In comparison to the sham operation group, the activity of calpain I was significantly increased in the control AF group (2.3 fold, p < 0.001), and decreased in the calpain inhibitor group (1.1 fold, p < 0.005). The calpain activity correlated with the apoptosis index (r = 0.9, p < 0.05). The apoptosis index was 1.0 +/- 0.2%, 11.8 +/- 6.8% and 3.5 +/- 2.1% in the sham operation group, control AF group and calpain inhibitor group, respectively. In the sham operation group, control AF group and calpain inhibitor group, the expressions of caspase-3 (13.0 +/- 1.9%, 52.8 +/- 4.3% and 33.6 +/- 3.7%), caspase-8 (40.1 +/- 5.3%, 92.6 +/- 6.5% and 55.3 +/- 5.9%), bcl-2 (65.8 +/- 6.1%, 52.0 +/- 5.7% and 69.9 +/- 5.3%) and ARC (70.2 +/- 8.6%, 68.8 +/- 7.3% and 81.5 +/- 8.8%) were calculated as immunohistochemical indexes, respectively. CONCLUSIONS: The calpain inhibitor N-Acetyl-Leu-Leu-Met attenuated apoptosis through a complicated network of apoptosis-related proteins, which may result in improvement of structural remodeling in atrial fibrillation.
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Apoptosis/efectos de los fármacos , Fibrilación Atrial/patología , Calpaína/antagonistas & inhibidores , Miocitos Cardíacos/efectos de los fármacos , Oligopéptidos/farmacología , Inhibidores de Proteasas/farmacología , Animales , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/metabolismo , Fibrilación Atrial/fisiopatología , Western Blotting , Peso Corporal/fisiología , Caspasa 3/metabolismo , Perros , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Contracción Miocárdica/efectos de los fármacos , Tamaño de los Órganos/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Angiogenesis is critical for tumor growth and metastasis. Tumor tissues induce the expression of angiogenesis-associated proteins on endothelial surface that can be targeted for tumor immunotherapy. In our study, the rat tumor endothelial proteins (EP) were isolated in situ via biotinylation of tumor vascular endothelial luminal surface followed by streptavidin affinity chromatography. The isolated tumor EP contained numerous up-regulated angiogenesis-associated endothelial proteins. The administration of these tumor EP as a vaccine to mice reduced the microvessel density in subcutaneous primary LLC tumors, delayed spontaneous LLC tumor metastasis and prolonged post-surgery life span. T lymphocytes from tumor EP-vaccinated mice lysed human umbilical vascular endothelial cells, but not tumor cells in vitro, in a dose-dependent manner. Furthermore, adoptive transfer of antitumor EP antibodies in vivo targeted to tumor endothelium and inhibited spontaneous LLC tumor metastasis. This study provides a successful preclinical exploration of the active immunotherapy for tumor by targeting tumor angiogenesis.
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Adenocarcinoma/irrigación sanguínea , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Endotelio Vascular/química , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Mamarias Experimentales/irrigación sanguínea , Proteínas de Neoplasias/uso terapéutico , Neovascularización Patológica/prevención & control , Traslado Adoptivo , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Biotinilación , Movimiento Celular , Electroforesis en Gel Bidimensional , Femenino , Citometría de Flujo , Immunoblotting , Técnicas para Inmunoenzimas , Inmunoglobulina G/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Conejos , Ratas , Ratas Endogámicas F344 , VacunaciónRESUMEN
Interleukin (IL)-13 is a key cytokine driving allergic and asthmatic responses and contributes to airway inflammation in cynomolgus monkeys after segmental challenge with Ascaris suum antigen. IL-13 bioactivity is mediated by a heterodimeric receptor (IL-13Ralpha1/IL-4Ralpha) and can be inhibited in vitro by targeting IL-13 interaction with either chain. However, in cytokine systems, in vitro neutralization activity may not always predict inhibitory function in vivo. To address the efficacy of two different IL-13 neutralization mechanisms in a primate model of atopic disease, two humanized monoclonal antibodies to IL-13 were generated, with highly homologous properties, differing in epitope recognition. Ab01 blocks IL-13 interaction with IL-4Ralpha, and Ab02 blocks IL-13 interaction with IL-13Ralpha1. In a cynomolgus monkey model of IgE responses to A. suum antigen, both Ab01 and Ab02 effectively reduced serum titers of Ascaris-specific IgE and diminished ex vivo Ascaris-triggered basophil histamine release, assayed 8 weeks after a single administration of antibody. The two antibodies also produced comparable reductions in pulmonary inflammation after lung segmental challenge with Ascaris antigen. Increased serum levels of IL-13, lacking demonstrable biological activity, were seen postchallenge in animals given either anti-IL-13 antibody but not in control animals given human IgG of irrelevant specificity. These findings demonstrate a potent effect of IL-13 neutralization on IgE-mediated atopic responses in a primate system and show that IL-13 can be efficiently neutralized by targeting either the IL-4Ralpha-binding epitope or the IL-13Ralpha1-binding epitope.
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Antígenos Helmínticos/inmunología , Ascaris/inmunología , Inmunoglobulina E/sangre , Inmunoglobulina G/inmunología , Inflamación/inmunología , Interleucina-13/inmunología , Pulmón/inmunología , Receptores de Interleucina-13/inmunología , Animales , Anticuerpos Antihelmínticos/inmunología , Basófilos/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Epítopos/inmunología , Liberación de Histamina/inmunología , Humanos , Macaca fascicularis , MasculinoRESUMEN
INTRODUCTION: The receptor for advanced glycation end products (RAGE), a multi-ligand member of the immunoglobulin superfamily, contributes to acute and chronic disease processes, including sepsis. METHODS: We studied the possible therapeutic role of RAGE inhibition in the cecal ligation and puncture (CLP) model of polymicrobial sepsis and a model of systemic listeriosis using mice genetically deficient in RAGE expression or mice injected with a rat anti-murine RAGE monoclonal antibody. RESULTS: The 7-day survival rates after CLP were 80% for RAGE-/- mice (n = 15) (P < 0.01 versus wild-type), 69% for RAGE+/- mice (n = 23), and 37% for wild-type mice (n = 27). Survival benefits were evident in BALB/c mice given anti-RAGE antibody (n = 15 per group) over serum-treated control animals (P < 0.05). Moreover, delayed treatment with anti-RAGE antibody up to 24 hours after CLP resulted in a significant survival benefit compared with control mice. There was no significant increase in tissue colony counts from enteric Gram-negative or Gram-positive bacteria in animals treated with anti-RAGE antibody. RAGE-/-, RAGE+/-, and anti-RAGE antibody-treated animals were resistant to lethality from Listeria monocytogenes by almost two orders of magnitude compared with wild-type mice. CONCLUSION: Further studies are warranted to determine the clinical utility of anti-RAGE antibody as a novel treatment for sepsis.
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Listeriosis/metabolismo , Listeriosis/terapia , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/biosíntesis , Sepsis/mortalidad , Sepsis/terapia , Animales , Anticuerpos Monoclonales/uso terapéutico , Modelos Animales de Enfermedad , Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Productos Finales de Glicación Avanzada/biosíntesis , Productos Finales de Glicación Avanzada/genética , Listeriosis/mortalidad , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Sepsis/genética , Tasa de Supervivencia , Síndrome de Respuesta Inflamatoria Sistémica/metabolismo , Síndrome de Respuesta Inflamatoria Sistémica/mortalidad , Síndrome de Respuesta Inflamatoria Sistémica/terapiaRESUMEN
The functional and structural alterations of vascular endothelium contribute to the initiation, progression, and complications of atherosclerotic plaque formation, but limited information is known about the molecular composition and pathways underlying pathological changes during atherosclerosis. We have developed an affinity proteomic strategy for in situ isolation and differential mapping of vascular endothelial proteins in normal and atherosclerotic aorta tissues. The selective labeling was carried out by perfusion of the blood vessels with an active biotin reagent for covalent modification of accessible vascular endothelial proteins. The biotinylated proteins were then enriched by streptavidin affinity chromatography, separated by SDS-PAGE, and subsequently characterized by LC-MS/MS. The described procedure led to the identification of 454 distinct proteins in normal and atherosclerotic aorta tissues. A majority of the proteins are plasma membrane associated and extracellular matrix proteins, and 81 showed altered expressions in atherosclerotic aorta tissue. The differentially expressed proteins are involved in immune and inflammatory responses, cell adhesion, and lipid metabolism. The method provides a new avenue for investigating the endothelial dysfunction and development of atherosclerosis.
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Aorta Torácica/química , Aterosclerosis/metabolismo , Endotelio Vascular/química , Perfusión , Proteínas/análisis , Proteómica , Animales , Aorta Torácica/metabolismo , Aorta Torácica/patología , Aterosclerosis/patología , Biotina , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas/clasificación , Proteínas/metabolismo , Coloración y EtiquetadoRESUMEN
BACKGROUND: Airway inflammation is a hallmark feature of asthma and a driver of airway hyperresponsiveness. IL-13 is a key inducer of airway inflammation in rodent models of respiratory disease, but a role for IL-13 has not been demonstrated in primates. OBJECTIVE: We sought to test the efficacy of a neutralizing antibody to human IL-13 in a cynomolgus monkey model of lung inflammation. METHODS: Using cynomolgus monkeys (Macaca fascicularis) that are sensitized to Ascaris suum through natural exposure, we developed a reproducible model of acute airway inflammation after segmental A suum antigen challenge. This model was used to test the in vivo efficacy of mAb13.2, a mouse mAb directed against human IL-13, and IMA-638, the humanized counterpart of mAb13.2. Bronchoalveolar lavage (BAL) cells and BAL fluid were collected before and after antigen challenge and assayed for cellular content by means of differential count. RESULTS: Total BAL cell count, eosinophil number, and neutrophil number were all reduced in animals treated with mAb13.2 or IMA-638 compared with values in control animals that were untreated, given saline, or treated with human IgG of irrelevant specificity. In addition, levels of eotaxin and RANTES in BAL fluid were reduced in anti-IL-13-treated animals compared with levels seen in control animals. CONCLUSION: These findings support a role for IL-13 in maintaining lung inflammation in response to allergen challenge in nonhuman primates. CLINICAL IMPLICATIONS: IL-13 neutralization with a specific antibody could be a useful therapeutic strategy for asthma.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Ascariasis/inmunología , Interleucina-13/antagonistas & inhibidores , Neumonía/inmunología , Neumonía/prevención & control , Secuencia de Aminoácidos , Animales , Anticuerpos Bloqueadores/uso terapéutico , Antígenos Helmínticos/inmunología , Ascaris suum , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Citocinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Interleucina-13/genética , Interleucina-13/inmunología , Macaca fascicularis , Masculino , Datos de Secuencia Molecular , Neumonía/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
IL-13 contributes to airway hyperresponsiveness, mucus secretion, inflammation, and fibrosis, suggesting that it plays a central role in asthma pathogenesis. Neutralization of IL-13 with sIL-13Ralpha2-Fc (sIL-13R) reduces allergen-induced airway responses in rodent models of respiratory disease, but its efficacy in a large animal model has not been previously reported. In this study, we determined whether two different strategies for IL-13 neutralization modified experimental asthma in sheep. Sheep with natural airway hypersensitivity to Ascaris suum antigen were treated intravenously either with sIL-13R, a strong antagonist of sheep IL-13 bioactivity in vitro, or with IMA-638 (IgG1, kappa), a humanized antibody to human IL-13. Higher doses of IMA-638 were used because, although it is a potent antagonist of human IL-13, this antibody has 20 to 30 times lower binding and neutralization activity against sheep IL-13. Control animals received human IgG of irrelevant specificity. Sheep were treated 24 h before inhalation challenge with nebulized A. suum. The effects on antigen-induced early and late bronchial responses, and antigen-induced hyperresponsiveness, were assessed. Both sIL-13R and IMA-638 provided dose-dependent inhibition of the antigen-induced late responses and airway hyperresponsiveness. The highest dose of IMA-638 also reduced the early phase response. These findings suggest that IL-13 contributes to allergen-induced airway responses in this sheep model of asthma, and that neutralization of IL-13 is an effective strategy for blocking these A. suum-induced effects.
Asunto(s)
Asma/tratamiento farmacológico , Asma/inmunología , Modelos Animales de Enfermedad , Interleucina-13/antagonistas & inhibidores , Interleucina-13/inmunología , Oveja Doméstica/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos/farmacología , Ascaris suum/fisiología , Asma/inducido químicamente , Asma/fisiopatología , Secuencia de Bases , Hiperreactividad Bronquial/parasitología , Hiperreactividad Bronquial/patología , Broncoconstricción/efectos de los fármacos , Broncoconstricción/inmunología , Carbacol/farmacología , Femenino , Células HT29 , Humanos , Interleucina-13/química , Interleucina-13/genética , Cinética , Datos de Secuencia Molecular , Pruebas de Neutralización , Receptores de Interleucina-13/metabolismo , Oveja Doméstica/parasitología , Solubilidad/efectos de los fármacos , Resonancia por Plasmón de Superficie , Factores de TiempoRESUMEN
We have previously demonstrated that a low dose of live myeloma FO cells induced a cellular immunity against tumor without additional modulating factors. In the present study, lyophilized myeloma FO cells were used to induce anti-tumor immunity. In a myeloma vaccination model, immunization with lyophilized myeloma FO cells alone induced a slight response. However, the immunity was dramatically enhanced by myeloma FO cells transfected with a recombinant adenovirus, Adv-1/GM-CSF. The immunocytochemical staining of Adv-1/GM-CSF transfected myeloma FO cells confirmed that more than 90% of cells were positive with GM-CSF expression. Results of sandwich ELISA showed the amount of secreted GM-CSF was 240 ng/24 h per 10(6) cells. Immunization with lyophilized myeloma FO cells secreting GM-CSF prevented the tumor growth in 60% of BALB/c mice. Antibodies against myeloma FO cells were found in the sera of immunized mice. Tumor-specific T-cell response was also evaluated using cytotoxic T lymphocyte assay. In conclusion, lyophilized myeloma FO cells secreting GM-CSF can be used as a potent vaccine to induce strong and protective anti-tumor immunity.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Inmunoterapia Adoptiva/métodos , Mieloma Múltiple/inmunología , Animales , Vacunas contra el Cáncer/genética , Línea Celular Tumoral , Femenino , Liofilización , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Mieloma Múltiple/terapia , TransfecciónRESUMEN
PURPOSE: Because tumor endothelium is rarely targeted by immunity but is critically important for tumor growth, the immunity against tumor endothelium is to be developed as a novel antitumor strategy. EXPERIMENTAL DESIGN: First, viable human umbilical vein endothelial cells (HUVEC) were immunized to C57BL/6 and BALB/c mice to evoke specific CTLs as well as antibodies against tumor endothelium. Lewis lung carcinoma or myeloma cells were subsequently inoculated to evaluate the effect on tumor growth by vaccination. Second, the effect on tumor metastasis by vaccination was studied using tumor-resected mice receiving HUVEC immunization 3 days after excision. Third, the immune sera and T lymphocytes from HUVEC-immunized mice were transferred to tumor-bearing mice and added to cultured HUVECs to investigate their antiproliferative effect. RESULTS: Viable HUVEC immunization showed potent antitumor effects in Lewis lung carcinoma and myeloma tumor models. Both immune sera and CTL inhibited tumor growth and specifically suppressed proliferation of HUVECs. Particularly, tumors entirely disappeared on day 90 after tumor inoculation in four of six tumor-bearing mice receiving CTL therapy. In a metastatic tumor model, we found that the HUVEC vaccination prolonged life span from 30.9 to 41.5 days after tumor resection compared with PBS-treated mice without apparent side effects. CONCLUSIONS: Vaccination with viable HUVECs evoked both humoral and cellular immunity against tumor microvasculature, and therefore significantly inhibited tumor growth and prolonged life span of tumor-resected mice. This may provide with a novel treatment for metastatic tumors. Moreover, we have established a convenient method to evoke specific CTL against tumor angiogenesis.
Asunto(s)
Carcinoma Pulmonar de Lewis/prevención & control , Endotelio Vascular/inmunología , Neoplasias Pulmonares/prevención & control , Melanoma Experimental/prevención & control , Neovascularización Patológica/inmunología , Vacunación , Animales , Formación de Anticuerpos , Antineoplásicos Hormonales/farmacología , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/inmunología , Humanos , Inmunidad Celular , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/inmunología , Masculino , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Metástasis de la Neoplasia , Tasa de Supervivencia , Linfocitos T/inmunología , Venas Umbilicales/citología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Th17 cells are a distinct lineage of effector CD4(+) T cells characterized by their production of interleukin (IL)-17. We demonstrate that Th17 cells also expressed IL-22, an IL-10 family member, at substantially higher amounts than T helper (Th)1 or Th2 cells. Similar to IL-17A, IL-22 expression was initiated by transforming growth factor beta signaling in the context of IL-6 and other proinflammatory cytokines. The subsequent expansion of IL-22-producing cells was dependent on IL-23. We further demonstrate that IL-22 was coexpressed in vitro and in vivo with both IL-17A and IL-17F. To study a functional relationship among these cytokines, we examined the expression of antimicrobial peptides by primary keratinocytes treated with combinations of IL-22, IL-17A, and IL-17F. IL-22 in conjunction with IL-17A or IL-17F synergistically induced the expression of beta-defensin 2 and S100A9 and additively enhanced the expression of S100A7 and S100A8. Collectively, we have identified IL-22 as a new cytokine expressed by Th17 cells that synergizes with IL-17A or IL-17F to regulate genes associated with skin innate immunity.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Inmunidad Innata/inmunología , Interleucina-17/inmunología , Interleucinas/inmunología , Transducción de Señal/inmunología , Animales , Péptidos Catiónicos Antimicrobianos/inmunología , Diferenciación Celular/inmunología , Interleucina-17/genética , Interleucinas/genética , Queratinocitos/metabolismo , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Factor de Crecimiento Transformador beta/metabolismo , Interleucina-22RESUMEN
Tyrosine kinase receptor B (TrkB) mediates neurotrophic effects of brain-derived neurotrophic factor (BDNF) to increase neuronal survival, differentiation, synaptic plasticity, and neurogenesis. The therapeutic potential of TrkB activation using BDNF has been demonstrated well in several preclinical models of CNS diseases, validating TrkB as a promising drug target. Therefore, we aimed to develop TrkB-specific receptor agonists by using a monoclonal antibody approach. After generation of hybridoma clones and assessment of their binding and functional activity, we identified five mouse monoclonal antibodies that show highly selective binding to TrkB and that induce robust activation of TrkB signaling. Epitope mapping studies using competition analysis showed that each of the monoclonal antibodies recognizes a unique binding site on TrkB, some of which are distinct from BDNF docking sites. These antibodies behave as true agonists based on their ability to both activate proximal and secondary signaling molecules downstream of TrkB receptors and promote neuronal survival and neurite outgrowth. The binding affinities and the functional efficacy of these antibodies are comparable to those of BDNF, whereas they do not bind to the p75 low-affinity neurotrophin receptor at all. Therefore, they could represent novel reagents to explore the pathophysiological roles of TrkB and its potential therapeutic utility in treating CNS disorders.