RESUMEN
Myeloid-derived suppressor cells (MDSCs) play a major role in cancer progression. In this study, we investigated the mechanisms by which complement C5a increases the capacity of polymorphonuclear MDSCs (PMN-MDSCs) to promote tumor growth and metastatic spread. Stimulation of PMN-MDSCs with C5a favored the invasion of cancer cells via a process dependent on the formation of neutrophil extracellular traps (NETs). NETosis was dependent on the production of high mobility group box 1 (HMGB1) by cancer cells. Moreover, C5a induced the surface expression of the HMGB1 receptors TLR4 and RAGE in PMN-MDSCs. In a mouse lung metastasis model, inhibition of C5a, C5a receptor-1 (C5aR1) or NETosis reduced the number of circulating-tumor cells (CTCs) and the metastatic burden. In support of the translational relevance of these findings, C5a was able to stimulate migration and NETosis in PMN-MDSCs obtained from lung cancer patients. Furthermore, myeloperoxidase (MPO)-DNA complexes, as markers of NETosis, were elevated in lung cancer patients and significantly correlated with C5a levels. In conclusion, C5a induces the formation of NETs from PMN-MDSCs in the presence of cancer cells, which may facilitate cancer cell dissemination and metastasis.
Asunto(s)
Complemento C5a/inmunología , Trampas Extracelulares/inmunología , Células Supresoras de Origen Mieloide/inmunología , Células Supresoras de Origen Mieloide/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Xenoinjertos , Humanos , Inmunofenotipificación , Ratones , Modelos Biológicos , Metástasis de la Neoplasia , Neoplasias/etiología , Neoplasias/metabolismo , Neoplasias/patología , Receptor de Anafilatoxina C5a/metabolismoRESUMEN
Singular blockade of C5a in experimental models of sepsis is known to confer protection by rescuing lethality and decreasing pro-inflammatory responses. However, the role of inhibiting C5a has not been evaluated in the context of sterile systemic inflammatory responses, like polytrauma and hemorrhagic shock (PT + HS). In our presented study, a novel and highly specific C5a L-aptamer, NoxD21, was used to block C5a activity in an experimental murine model of PT + HS. The aim of the study was to assess early modulation of inflammatory responses and lung damage 4 h after PT + HS induction. NoxD21-treated PT + HS mice displayed greater polymorphonuclear cell recruitment in the lung, increased pro-inflammatory cytokine levels in the bronchoalveolar lavage fluids (BALF) and reduced myeloperoxidase levels within the lung tissue. An in vitro model of the alveolar-capillary barrier was established to confirm these in vivo observations. Treatment with a polytrauma cocktail induced barrier damage only after 16 h, and NoxD21 treatment in vitro did not rescue this effect. Furthermore, to test the exact role of both the cognate receptors of C5a (C5aR1 and C5aR2), experimental PT + HS was induced in C5aR1 knockout (C5aR1 KO) and C5aR2 KO mice. Following 4 h of PT + HS, C5aR2 KO mice had significantly reduced IL-6 and IL-17 levels in the BALF without significant lung damage, and both, C5aR1 KO and C5aR2 KO PT + HS animals displayed reduced MPO levels within the lungs. In conclusion, the C5aR2 could be a putative driver of early local inflammatory responses in the lung after PT + HS.
Asunto(s)
Complemento C5a/metabolismo , Inflamación/patología , Pulmón/patología , Traumatismo Múltiple/complicaciones , Traumatismo Múltiple/metabolismo , Receptor de Anafilatoxina C5a/metabolismo , Choque Hemorrágico/complicaciones , Choque Hemorrágico/metabolismo , Adulto , Animales , Aptámeros de Péptidos/farmacología , Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar , Capilares/patología , Línea Celular , Quimiotaxis/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Alveolos Pulmonares/patología , Receptor de Anafilatoxina C5a/deficienciaRESUMEN
BACKGROUND: Community-acquired pneumonia and associated sepsis cause high mortality despite antibiotic treatment. Uncontrolled inflammatory host responses contribute to the unfavorable outcome by driving lung and extrapulmonary organ failure. The complement fragment C5a holds significant proinflammatory functions and is associated with tissue damage in various inflammatory conditions. The authors hypothesized that C5a concentrations are increased in pneumonia and C5a neutralization promotes barrier stabilization in the lung and is protective in pneumococcal pulmonary sepsis. METHODS: The authors investigated regulation of C5a in pneumonia in a prospective patient cohort and in experimental pneumonia. Two complementary models of murine pneumococcal pneumonia were applied. Female mice were treated with NOX-D19, a C5a-neutralizing L-RNA-aptamer. Lung, liver, and kidney injury and the inflammatory response were assessed by measuring pulmonary permeability (primary outcome), pulmonary and blood leukocytes, cytokine concentrations in lung and blood, and bacterial load in lung, spleen, and blood, and performing histologic analyses of tissue damage, apoptosis, and fibrin deposition (n = 5 to 13). RESULTS: In hospitalized patients with pneumonia (n = 395), higher serum C5a concentrations were observed compared to healthy subjects (n = 24; 6.3 nmol/l [3.9 to 10.0] vs. 4.5 nmol/l [3.8 to 6.6], median [25 to 75% interquartile range]; difference: 1.4 [95% CI, 0.1 to 2.9]; P = 0.029). Neutralization of C5a in mice resulted in lower pulmonary permeability in pneumococcal pneumonia (1.38 ± 0.89 vs. 3.29 ± 2.34, mean ± SD; difference: 1.90 [95% CI, 0.15 to 3.66]; P = 0.035; n = 10 or 11) or combined severe pneumonia and mechanical ventilation (2.56 ± 1.17 vs. 7.31 ± 5.22; difference: 4.76 [95% CI, 1.22 to 8.30]; P = 0.011; n = 9 or 10). Further, C5a neutralization led to lower blood granulocyte colony-stimulating factor concentrations and protected against sepsis-associated liver injury. CONCLUSIONS: Systemic C5a is elevated in pneumonia patients. Neutralizing C5a protected against lung and liver injury in pneumococcal pneumonia in mice. Early neutralization of C5a might be a promising adjunctive treatment strategy to improve outcome in community-acquired pneumonia.
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Aptámeros de Nucleótidos/administración & dosificación , Complemento C5a/antagonistas & inhibidores , Neumonía Neumocócica/sangre , Neumonía Neumocócica/prevención & control , Sepsis/sangre , Sepsis/prevención & control , Animales , Anticuerpos Neutralizantes/administración & dosificación , Biomarcadores/sangre , Estudios de Cohortes , Complemento C5a/metabolismo , Femenino , Factores Inmunológicos/antagonistas & inhibidores , Factores Inmunológicos/sangre , Ratones , Ratones Endogámicos C57BL , Estudios ProspectivosRESUMEN
Microvascular injury during acute rejection has been associated with massive infiltration of CD4+ T effector cells, and the formation of complement products (C3a and C5a). Regulatory T cells (Tregs) are potent immunosuppressors of the adaptive immune system and have proven sufficient to rescue microvascular impairments. Targeting C5a has been linked with improved microvascular recovery, but its effects on the Treg and T effector balance is less well known. Here, we demonstrate the impact of C5a blockade on Treg induction and microvascular restoration in rejecting mouse airway allografts. BALB/câC57BL/6 allografts were treated with a C5a-neutralizing l-aptamer (10 mg/kg, i.p. at d0 and every second day thereafter), and allografts were serially monitored for Treg infiltration, tissue oxygenation (tpO2), microvascular blood flow, and functional microvasculature between donor and recipients during allograft rejection. We demonstrated that C5a blocking significantly leads to enhanced presence of Tregs in the allograft, reinstates donor-recipient functional microvasculature, improves tpO2, microvascular blood flow, and epithelial repair, followed by an upregulation of IL-5, TGF-ß, IL-10 vascular endothelial growth factor, and ANGPT1 gene expression, while it maintained a healthy epithelium and prevented subepithelial collagen deposition at d28 posttransplantation. Together, these data indicate that inhibition of C5a signaling has potential to preserve microvasculature and rescue allograft from a sustained hypoxic/ischemic phase, limits airway tissue remodeling through the induction of Treg-mediated immune tolerance. These findings may be useful in designing anti-C5a therapy in combination with existing immunosuppressive regimens to rescue tissue/organ rejection.
Asunto(s)
Complemento C5a/antagonistas & inhibidores , Células Epiteliales/inmunología , Células Epiteliales/patología , Rechazo de Injerto/prevención & control , Linfocitos T Reguladores/inmunología , Tráquea/trasplante , Aloinjertos/inmunología , Animales , Complemento C5a/inmunología , Rechazo de Injerto/inmunología , Tolerancia Inmunológica , Inmunosupresores/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microvasos/inmunología , Sistema Respiratorio/citología , Sistema Respiratorio/inmunología , Tráquea/inmunología , Trasplante HomólogoRESUMEN
BACKGROUND: Calcitonin gene-related peptide (CGRP) plays a major role in the pathogenesis of migraine and other primary headaches. Spinal trigeminal neurons integrate nociceptive afferent input from trigeminal tissues including intracranial afferents, and their activity is thought to reflect facial pain and headache in man. CGRP receptor inhibitors and anti-CGRP antibodies have been demonstrated to be therapeutically effective in migraine. In parallel, CGRP receptor inhibition has been shown to lower spinal trigeminal neuron activity in animal models of meningeal nociception. METHODS: In a rat model of meningeal nociception, single cell activity of neurons in the spinal trigeminal nucleus with meningeal afferent input was recorded to test a further pharmacological approach, scavenging CGRP with a CGRP-binding L-RNA oligonucleotide, the L-aptamer NOX-C89. Cumulative ascending doses of NOX-C89 were intravenously infused. RESULTS: Spontaneous activity of spinal trigeminal neurons did not change after 0.05 mg/kg NOX-C89, however, after additional infusion of 0.5 mg/kg and 5 mg/kg NOX-C89, spontaneous activity was dose-dependently reduced. Identical doses of a control L-aptamer had no effect. This pharmacological effect of NOX-C89 was observed 10-25 min after infusion, but no difference was detected in the period 0-5 min. For comparison, the previously investigated CGRP receptor antagonist olcegepant had reduced activity within 5 min after infusion. Alongside the reduced spontaneous activity, after infusion of NOX-C89 the heat-induced neuronal activity was abolished. CONCLUSIONS: Scavenging CGRP by mirror-image RNA aptamers provides further evidence that this approach can be used to control spinal trigeminal activity.
Asunto(s)
Aptámeros de Nucleótidos/farmacología , Péptido Relacionado con Gen de Calcitonina/metabolismo , Meninges , Trastornos Migrañosos/tratamiento farmacológico , Neuronas/efectos de los fármacos , Nocicepción/efectos de los fármacos , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , Núcleo Espinal del Trigémino/efectos de los fármacos , Animales , Aptámeros de Nucleótidos/administración & dosificación , Modelos Animales de Enfermedad , Masculino , ARN , Ratas , Ratas WistarRESUMEN
Immune checkpoint inhibitors promote T cell-mediated killing of cancer cells; however, only a subset of patients benefit from the treatment. A possible reason for this limitation may be that the tumor microenvironment (TME) is immune privileged, which may exclude cytotoxic T cells from the vicinity of cancer cells. The chemokine CXCL12 is key to the TME-driven immune suppression. In this study, we investigated the potential of CXCL12 inhibition by use of the clinical-stage l-RNA-aptamer NOX-A12 (olaptesed pegol) to increase the number of tumor-infiltrating lymphocytes. We used heterotypic tumor-stroma spheroids that mimic a solid tumor with a CXCL12-abundant TME. NOX-A12 enhanced the infiltration of T and NK cells in a dose-dependent manner. NOX-A12 and PD-1 checkpoint inhibition synergistically activated T cells in the spheroids, indicating that the agents complement each other. The findings were validated in vivo in a syngeneic murine model of colorectal cancer in which the addition of NOX-A12 improved anti-PD-1 therapy. Taken together, our work shows that CXCL12 inhibition can break the immune-privileged status of the TME by paving the way for immune effector cells to enter into the tumor, thereby broadening the applicability of checkpoint inhibitors in cancer patients. Cancer Immunol Res; 5(11); 950-6. ©2017 AACR.
Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Aptámeros de Nucleótidos/uso terapéutico , Quimiocina CXCL12/antagonistas & inhibidores , Neoplasias del Colon/tratamiento farmacológico , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Microambiente Tumoral/efectos de los fármacos , Animales , Antineoplásicos Inmunológicos/farmacología , Aptámeros de Nucleótidos/farmacología , Línea Celular Tumoral , Quimiocina CXCL12/inmunología , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Ratones Endogámicos BALB C , Microambiente Tumoral/inmunologíaRESUMEN
Several mechanisms have been postulated for orchestrating the mobilization of hematopoietic stem/progenitor cells (HSPCs), and we previously proposed that activation of the complement cascade plays a crucial role in the initiation and execution of the egress of HSPCs from bone marrow (BM) into peripheral blood (PB). In support of this notion, we demonstrated that mice deficient in the mannan-binding lectin (MBL) pathway, which activates the proximal part of the complement cascade, as well as mice deficient in the fifth component of the complement cascade (C5), which is part of the distal part of the complement cascade, are poor mobilizers. To further narrow down on the exact mechanisms and the molecules involved, we performed studies in mice that do not express the receptor C5aR, which binds the C5 cleavage fragments, C5a and C5adesArg. We also employed the plasma stable nucleic acid aptamer AON-D21 that binds and neutralizes C5a and C5adesArg. We present evidence that mice deficient in C5aR or treated with AON-D21 are poor HSPC mobilizers, thereby establishing a critical role for the C5a/C5adesArg-C5aR axis in the mobilization process. While enhancing mobilization is of clinical importance for poor mobilizers, inhibition of the complement cascade could be of therapeutic importance in patients suffering from paroxysmal nocturnal hemoglobinuria (PNH) or acquired hemolytic syndrome (aHUS).
Asunto(s)
Complemento C5a/genética , Células Madre Hematopoyéticas/citología , Lectina de Unión a Manosa/genética , Receptor de Anafilatoxina C5a/genética , Anafilatoxinas/genética , Animales , Activación de Complemento/genética , Complemento C5a des-Arginina/genética , Lectina de Unión a Manosa de la Vía del Complemento/genética , Movilización de Célula Madre Hematopoyética/métodos , Células Madre Hematopoyéticas/metabolismo , Hemoglobinuria Paroxística , Humanos , Lectina de Unión a Manosa/deficiencia , RatonesRESUMEN
Disruption of the programmed cell death protein 1 (PD-1) pathway with immune checkpoint inhibitors represents a major breakthrough in the treatment of non-small cell lung cancer. We hypothesized that combined inhibition of C5a/C5aR1 and PD-1 signaling may have a synergistic antitumor effect. The RMP1-14 antibody was used to block PD-1, and an L-aptamer was used to inhibit signaling of complement C5a with its receptors. Using syngeneic models of lung cancer, we demonstrate that the combination of C5a and PD-1 blockade markedly reduces tumor growth and metastasis and leads to prolonged survival. This effect is accompanied by a negative association between the frequency of CD8 T cells and myeloid-derived suppressor cells within tumors, which may result in a more complete reversal of CD8 T-cell exhaustion. Our study provides support for the clinical evaluation of anti-PD-1 and anti-C5a drugs as a novel combination therapeutic strategy for lung cancer.Significance: Using a variety of preclinical models of lung cancer, we demonstrate that the blockade of C5a results in a substantial improvement in the efficacy of anti-PD-1 antibodies against lung cancer growth and metastasis. This study provides the preclinical rationale for the combined blockade of PD-1/PD-L1 and C5a to restore antitumor immune responses, inhibit tumor cell growth, and improve outcomes of patients with lung cancer. Cancer Discov; 7(7); 694-703. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 653.
Asunto(s)
Antígeno B7-H1/antagonistas & inhibidores , Complemento C5a/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Aptámeros de Péptidos/inmunología , Aptámeros de Péptidos/uso terapéutico , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Proliferación Celular/efectos de los fármacos , Complemento C5a/inmunología , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Células Supresoras de Origen Mieloide/efectos de los fármacos , Células Supresoras de Origen Mieloide/inmunología , Metástasis de la Neoplasia , Receptor de Muerte Celular Programada 1/inmunología , Transducción de SeñalRESUMEN
Inflammatory events occurring in dystrophic muscles contribute to the progression of Duchenne muscular dystrophy (DMD). Low-intensity training (LIT) attenuates the phenotype of mdx mice, an animal model for DMD. Therefore, we postulated that LIT could have anti-inflammatory properties. We assessed levels of inflammatory cytokines and infiltrated immune cells in gastrocnemius muscle of mdx mice after LIT. We detected high levels of complement component C5a, chemokine ligand (CCL) 2, CD68+ monocytes/macrophages, and proinflammatory M1 macrophages in muscles of mdx mice. LIT decreased CCL2 levels, increased CD68+ cell numbers, and shifted the macrophage population to the regenerative M2 type. We investigated whether inhibition of C5a or CCL2 with L-aptamers could mimic the effects of LIT. Although no effect of CCL2 inhibition was detected, treatment with the C5a inhibitor, NOX-D21, rescued the phenotype of nonexercised mdx mice, but not of exercised ones. In both cases, the level of CD68+ cells increased and macrophage populations leaned toward the inflammatory M1 type. In muscles of nonexercised treated mice, the level of IL-1 receptor antagonist increased, damage decreased, and fibers were switched toward the glycolytic fast type; in muscles of exercised mice, fibers were switched to the oxidative slow type. These results reveal the effects of LIT on the inflammatory status of mdx mice and suggest that NOX-D21 could be an anti-inflammatory drug for DMD.
Asunto(s)
Complemento C5a/antagonistas & inhibidores , Distrofia Muscular Animal/metabolismo , Condicionamiento Físico Animal/fisiología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Aptámeros de Nucleótidos/farmacología , Quimiocina CCL2/antagonistas & inhibidores , Citocinas/metabolismo , Modelos Animales de Enfermedad , Metabolismo Energético/fisiología , Miembro Anterior , Macrófagos/fisiología , Masculino , Ratones Endogámicos mdx , Fuerza Muscular/fisiología , Músculo Esquelético/fisiología , Distrofia Muscular Animal/fisiopatología , Miositis/fisiopatología , Miositis/prevención & control , Fenotipo , Natación/fisiologíaRESUMEN
Mobilized bone marrow-derived stem cells (BMSC) have been discussed as an alternative strategy for endogenous repair. Thereby, different approaches for BMSC mobilization have been pursued. Herein, the role of a newly discovered oligonucleotide for retinal homing and regeneration capability of BMSCs was investigated in the sodium iodate (NaIO3) model of retinal degeneration. Mobilization was achieved in GFP-chimera with NOX-A12, a CXC-motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 (SDF-1)-neutralizing L-aptamer. BMSC homing was directed by intravitreal SDF-1 injection. Visual acuity was measured using the optokinetic reflex. Paraffin cross sections were stained with hematoxylin and eosin for retinal thickness measurements. Immunohistochemistry was performed to investigate the expression of cell-specific markers after mobilization. A single dose of NOX-A12 induced significant mobilization of GFP+ cells which were found in all layers within the degenerating retina. An additional intravitreal injection of SDF-1 increased migration towards the site of injury. Thereby, the number of BMSCs (Sca-1+) found in the damaged retina increased whereas a decrease of activated microglia (Iba-1+) was found. The mobilization led to significantly increased visual acuity. However, no significant changes in retinal thickness or differentiation towards retinal cell types were detected. Systemic mobilization by a single dose of NOX-A12 showed increased homing of BMSCs into the degenerated retina, which was associated with improved visual function when injection of SDF-1 was additionally performed. The redistribution of the cells to the site of injury combined with their observed beneficial effects support the endogenous therapeutic strategy for retinal repair.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Quimiocina CXCL12/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Degeneración Retiniana/prevención & control , Animales , Aptámeros de Nucleótidos , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Quimiocina CXCL12/administración & dosificación , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Inyecciones Intraoculares , Yodatos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Retina/citología , Degeneración Retiniana/inducido químicamente , Degeneración Retiniana/fisiopatología , Tubulina (Proteína)/metabolismo , Agudeza Visual/efectos de los fármacosRESUMEN
PURPOSE: To investigate the influence of complement component C5a inhibition on laser-induced choroidal neovascularization (CNV) in mice using a C5a specific L-aptamer. METHODS: In C57BL/6 J mice CNV was induced by argon-laser, C5a-inhibitor (NOX-D20) was intravitreally injected in three concentrations: 0.3, 3.0, and 30 mg/ml. The unPEGylated derivate (NOX-D20001) was applied at 3.0 mg/ml; the vehicle (5 % glucose) was injected in controls. Vascular leakage was evaluated using fluorescence angiography, CNV area was examined immunohistochemically. Activated immune cells surrounding the CNV lesion and potential cytotoxicity were analyzed. RESULTS: Compared to controls, CNV areas were significantly reduced after NOX-D20 injection at a concentration of 0.3 and 3.0 mg/ml (p = 0.042; p = 0.016). NOX-D20001 significantly decreased CNV leakage but not the area (p = 0.007; p = 0.276). At a concentration of 30 mg/ml, NOX-D20 did not reveal significant effects on vascular leakage or CNV area (p = 0.624; p = 0.121). The amount of CD11b positive cells was significantly reduced after treatment with 0.3 and 3.0 mg/ml NOX-D20 (p = 0.027; p = 0.002). No adverse glial cell proliferation or increased apoptosis were observed at effective dosages. CONCLUSIONS: Our findings demonstrate that the targeted inhibition of complement component C5a reduces vascular leakage and neovascular area in laser-induced CNV in mice. NOX-D20 was proven to be an effective and safe agent that might be considered as a therapeutic candidate for CNV treatment. The deficiency of activated immune cells highlights promising new aspects in the pathology of choroidal neovascularization, and warrants further investigations.
Asunto(s)
Aptámeros de Nucleótidos/uso terapéutico , Neovascularización Coroidal/tratamiento farmacológico , Complemento C5a/antagonistas & inhibidores , Serina Endopeptidasas/uso terapéutico , Animales , Apoptosis , Aptámeros de Nucleótidos/efectos adversos , Permeabilidad Capilar/efectos de los fármacos , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Angiografía con Fluoresceína , Células Gigantes/patología , Inmunohistoquímica , Inyecciones Intravítreas , Leucocitos/patología , Ratones , Ratones Endogámicos C57BL , Serina Endopeptidasas/efectos adversos , Cuerpo Vítreo/metabolismoRESUMEN
L-Oligonucleotide aptamers (Spiegelmers) consist of non-natural L-configured nucleotides and are of particular therapeutic interest due to their high resistance to plasma nucleases. The anaphylatoxin C5a, a potent inflammatory mediator generated during complement activation that has been implicated with organ damage, can be efficiently targeted by Spiegelmers. Here, we present the first crystallographic structures of an active Spiegelmer, NOX-D20, bound to its physiological targets, mouse C5a and C5a-desArg. The structures reveal a complex 3D architecture for the L-aptamer that wraps around C5a, including an intramolecular G-quadruplex stabilized by a central Ca(2+) ion. Functional validation of the observed L-aptamer:C5a binding mode through mutational studies also rationalizes the specificity of NOX-D20 for mouse and human C5a against macaque and rat C5a. Finally, our structural model provides the molecular basis for the Spiegelmer affinity improvement through positional L-ribonucleotide to L-deoxyribonucleotide exchanges and for its inhibition of the C5a:C5aR interaction.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Complemento C5a des-Arginina/metabolismo , Complemento C5a/metabolismo , ADN/metabolismo , ARN/metabolismo , Anafilatoxinas , Animales , Aptámeros de Nucleótidos/química , Calcio , Línea Celular , Ensayos de Migración Celular , Complemento C5a/química , Complemento C5a des-Arginina/química , Cristalografía por Rayos X , ADN/química , Escherichia coli , Humanos , Macaca , Ratones , Conformación de Ácido Nucleico , ARN/química , Ratas , Receptor de Anafilatoxina C5a/metabolismo , Proteínas Recombinantes , Técnica SELEX de Producción de AptámerosRESUMEN
Inflammation may contribute to disease progression in arrhythmogenic cardiomyopathy (ACM). However, its role in this process is unresolved. Our goal was to delineate the pathogenic role of the complement system in a new animal model of ACM and in human disease. Using cardiac histology, echocardiography, and electrocardiography, we have demonstrated that the desmin-null mouse (Des-/-) recapitulates most of the pathognomonic features of human ACM. Massive complement activation was observed in the Des-/- myocardium in areas of necrotic cells debris and inflammatory infiltrate. Analysis of C5aR-/-Des-/- double-null animals and a pharmaceutical approach using a C5a inhibitor were used to delineate the pathogenic role of the complement system in the disease progression. Our findings indicate that inhibiting C5aR (CD88) signaling improves cardiac function, histopathology, arrhythmias, and survival after endurance. Containment of the inflammatory reaction at the initiation of cardiac tissue injury (2-3 weeks of age), with consequently reduced myocardial remodeling and the absence of a direct long-lasting detrimental effect of C5a-C5aR signaling on cardiomyocytes, could explain the beneficial action of C5aR ablation in Des-/- cardiomyopathy. We extend the relevance of these findings to human pathophysiology by showing for the first time significant complement activation in the cardiac tissues of patients with ACM, thus suggesting that complement modulation could be a new therapeutic target for ACM.
Asunto(s)
Cardiomiopatías/inmunología , Complemento C5a/inmunología , Receptores de Complemento/inmunología , Adulto , Animales , Arritmias Cardíacas/inmunología , Arritmias Cardíacas/patología , Western Blotting , Cardiomiopatías/patología , Desmina/deficiencia , Modelos Animales de Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Etiquetado Corte-Fin in Situ , Inflamación/inmunología , Inflamación/patología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana EdadRESUMEN
Spiegelmers are synthetic target-binding oligonucleotides built from non-natural l-nucleotides. Like aptamers, Spiegelmers fold into distinct shapes that bind the targets with high affinity and selectivity. Furthermore, the mirror-image configuration confers plasma stability and immunological passivity. Various Spiegelmers against pharmacologically attractive targets were shown to be efficacious in animal models. Three Spiegelmer candidates: emapticap pegol (NOX-E36; anti-CCL2), olaptesed pegol (NOX-A12; anti-CXCL12) and lexaptepid pegol (NOX-H94; anti-hepcidin), underwent regulatory safety studies, demonstrated good safety profiles in healthy volunteers and were taken into Phase IIa studies in patients. Proof-of-concept for emapticap pegol has recently been demonstrated in diabetic nephropathy patients. Furthermore, promising interim Phase IIa data of olaptesed pegol and lexapteptid pegol also suggest efficacy in the respective patient populations.
Asunto(s)
Aptámeros de Nucleótidos , Animales , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacocinética , Aptámeros de Nucleótidos/farmacología , Aptámeros de Nucleótidos/uso terapéutico , Modelos Animales de Enfermedad , Medicamentos Genéricos , HumanosRESUMEN
Bone marrow (BM) metastasis remains one of the main causes of death associated with solid tumors as well as multiple myeloma (MM). Targeting the BM niche to prevent or modulate metastasis has not been successful to date. Here, we show that stromal cell-derived factor-1 (SDF-1/CXCL12) is highly expressed in active MM, as well as in BM sites of tumor metastasis and report on the discovery of the high-affinity anti-SDF-1 PEGylated mirror-image l-oligonucleotide (olaptesed-pegol). In vivo confocal imaging showed that SDF-1 levels are increased within MM cell-colonized BM areas. Using in vivo murine and xenograft mouse models, we document that in vivo SDF-1 neutralization within BM niches leads to a microenvironment that is less receptive for MM cells and reduces MM cell homing and growth, thereby inhibiting MM disease progression. Targeting of SDF-1 represents a valid strategy for preventing or disrupting colonization of the BM by MM cells.
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Médula Ósea/patología , Quimiocina CXCL12/antagonistas & inhibidores , Mieloma Múltiple/terapia , Oligonucleótidos/farmacología , Animales , Médula Ósea/metabolismo , Neoplasias de la Médula Ósea/metabolismo , Neoplasias de la Médula Ósea/secundario , Ácidos Borónicos/farmacología , Bortezomib , Quimiocina CXCL12/biosíntesis , Quimiocina CXCL12/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Ratones SCID , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Metástasis de la Neoplasia , Oligonucleótidos/química , Oligonucleótidos/genética , Polietilenglicoles/química , Pirazinas/farmacologíaRESUMEN
The sphingolipid S1P (sphingosine 1-phosphate) is known to be involved in a number of pathophysiological conditions such as cancer, autoimmune diseases and fibrosis. It acts extracellularly through a set of five G-protein-coupled receptors, but its intracellular actions are also well documented. Employing in vitro selection techniques, we identified an L-aptamer (Spiegelmer®) to S1P designated NOX-S93. The binding affinity of NOX-S93 to S1P had a Kd value of 4.3 nM. The Spiegelmer® shows equal binding to dihydro-S1P, but no cross-reactivity to the related lipids sphingosine, lysophosphatidic acid, ceramide, ceramide-1-phosphate or sphingosine phosphocholine. In stably transfected CHO (Chinese-hamster ovary) cell lines expressing the S1P receptors S1PR1 or S1PR3, NOX-S93 inhibits S1P-mediated ß-arrestin recruitment and intracellular calcium release respectively, with IC50 values in the low nanomolar range. The pro-angiogenic activity of S1P, and of the growth factors VEGF-A (vascular endothelial growth factor-A), FGF-2 (fibroblast growth factor-2) and IGF-1 (insulin-like growth factor-1), was effectively blocked by NOX-S93 in a cellular angiogenesis assay employing primary human endothelial cells. These data provide further evidence for the relevance of extracellular S1P as a central mediator of angiogenesis, suggesting pharmacological S1P neutralization as a promising treatment alternative to current anti-angiogenesis approaches.
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Inhibidores de la Angiogénesis/aislamiento & purificación , Aptámeros de Nucleótidos/farmacología , Lisofosfolípidos/antagonistas & inhibidores , Esfingosina/análogos & derivados , Inhibidores de la Angiogénesis/farmacología , Animales , Arrestinas/metabolismo , Células CHO , Calcio/metabolismo , Cricetulus , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Humanos , Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Neovascularización Patológica , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , beta-ArrestinasRESUMEN
An in vivo near-infrared fluorescence (NIRF) imaging technique is described for therapy monitoring of ankle joints affected by collagen-induced arthritis, a model of human rheumatoid arthritis. Arthritis was induced in rats by intradermal injections of collagen and Freund's incomplete adjuvant. For in vivo imaging, the nonspecific NIR dye tetrasulfocyanine (TSC) was used. Prior to and after treatment with a nonsteroidal anti-inflammatory drug, meloxicam, or analgesic drug, tramadol hydrochloride (which served as no-therapy control), normalized fluorescence intensities of each ankle joint were measured. Additionally, each ankle joint was characterized by clinical arthritis scoring and histopathology. Over a 3-week treatment period, a significant difference in disease progression between animals treated with meloxicam and tramadol hydrochloride was detected. A statistically significant improvement in ankle joint pathology from high- or moderate-grade to moderate- or low-grade upon meloxicam therapy, as determined by clinical evaluation, translated into a significant decrease in fluorescence intensity. In contrast, all arthritic joints of the no-therapy control group deteriorated to high-grade arthritis with high-fluorescence intensities in NIRF imaging.
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Artritis Experimental , Artritis Reumatoide , Imagen Óptica/métodos , Animales , Antiinflamatorios/uso terapéutico , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/patología , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/patología , Femenino , Colorantes Fluorescentes , Indoles , RatasRESUMEN
BACKGROUND: Development of alcohol dependence, a chronic and relapsing disease, largely depends on the effects of alcohol on the brain reward systems. By elucidating the mechanisms involved in alcohol use disorder, novel treatment strategies may be developed. Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor 1A, acts as an important regulator of energy balance. Recently ghrelin and its receptor were shown to mediate alcohol reward and to control alcohol consumption in rodents. However, the role of central versus peripheral ghrelin for alcohol reward needs to be elucidated. METHODS: Given that ghrelin mainly is produced by peripheral organs, the present study was designed to investigate the role of circulating endogenous ghelin for alcohol reward and for alcohol intake in rodents. RESULTS: We showed that the Spiegelmer NOX-B11-2, which binds and neutralizes acylated ghrelin in the periphery with high affinity and thus prevents its brain access, does not attenuate the alcohol-induced locomotor activity, accumbal dopamine release and expression of conditioned place preference in mice. Moreover, NOX-B11-2 does not affect alcohol intake using the intermittent access 20% alcohol 2-bottle-choice drinking paradigm in rats, suggesting that circulating ghrelin does not regulate alcohol intake or the rewarding properties of alcohol. In the present study, we showed however, that NOX-B11-2 reduced food intake in rats supporting a role for circulating ghrelin as physiological regulators of food intake. Moreover, NOX-B11-2 did not affect the blood alcohol concentration in mice. CONCLUSIONS: Collectively, the past and present studies suggest that central, rather than peripheral, ghrelin signaling may be a potential target for pharmacological treatment of alcohol dependence.
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Consumo de Bebidas Alcohólicas/sangre , Ghrelina/sangre , Recompensa , Consumo de Bebidas Alcohólicas/fisiopatología , Animales , Biomarcadores/sangre , Ghrelina/antagonistas & inhibidores , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Oligonucleótidos/farmacología , Ratas , Ratas WistarRESUMEN
Complement factor C5a is a potent proinflammatory mediator that contributes to the pathogenesis of numerous inflammatory diseases. Here, we describe the discovery of NOX-D20, a PEGylated biostable mirror-image mixed (l-)RNA/DNA aptamer (Spiegelmer) that binds to mouse and human C5a with picomolar affinity. In vitro, NOX-D20 inhibited C5a-induced chemotaxis of a CD88-expressing cell line and efficiently antagonized the activation of primary human polymorphonuclear leukocytes (PMN) by C5a. Binding of NOX-D20 to the C5a moiety of human C5 did not interfere with the formation of the terminal membrane attack complex (MAC). In sepsis, for which a specific interventional therapy is currently lacking, complement activation and elevated levels of C5a are suggested to contribute to multiorgan failure and mortality. In the model of polymicrobial sepsis induced by cecal ligation and puncture (CLP), NOX-D20 attenuated inflammation and organ damage, prevented the breakdown of the vascular endothelial barrier, and improved survival. Our study suggests NOX-D20 as a new therapeutic candidate for the treatment of sepsis.
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Antiinflamatorios no Esteroideos/uso terapéutico , Aptámeros de Nucleótidos/uso terapéutico , Complemento C5a/antagonistas & inhibidores , Insuficiencia Multiorgánica/prevención & control , Peritonitis/tratamiento farmacológico , Sepsis/tratamiento farmacológico , Animales , Antiinflamatorios no Esteroideos/farmacocinética , Aptámeros de Nucleótidos/farmacocinética , Complemento C5a/inmunología , Complemento C5a/metabolismo , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Insuficiencia Multiorgánica/tratamiento farmacológico , Neutrófilos/inmunología , Neutrófilos/metabolismo , Peritonitis/complicaciones , Peritonitis/fisiopatología , Sepsis/complicacionesRESUMEN
Excessive secretion of glucagon, a functional insulin antagonist, significantly contributes to hyperglycemia in type 1 and type 2 diabetes. Accordingly, immunoneutralization of glucagon or genetic deletion of the glucagon receptor improved glucose homeostasis in animal models of diabetes. Despite this strong evidence, agents that selectively interfere with endogenous glucagon have not been implemented in clinical practice yet. We report the discovery of mirror-image DNA-aptamers (Spiegelmer®) that bind and inhibit glucagon. The affinity of the best binding DNA oligonucleotide was remarkably increased (>25-fold) by the introduction of oxygen atoms at selected 2'-positions through deoxyribo- to ribonucleotide exchanges resulting in a mixed DNA/RNA-Spiegelmer (NOX-G15) that binds glucagon with a Kd of 3 nm. NOX-G15 shows no cross-reactivity with related peptides such as glucagon-like peptide-1, glucagon-like peptide-2, gastric-inhibitory peptide, and prepro-vasoactive intestinal peptide. In vitro, NOX-G15 inhibits glucagon-stimulated cAMP production in CHO cells overexpressing the human glucagon receptor with an IC50 of 3.4 nm. A single injection of NOX-G15 ameliorated glucose excursions in intraperitoneal glucose tolerance tests in mice with streptozotocin-induced (type 1) diabetes and in a non-genetic mouse model of type 2 diabetes. In conclusion, the data suggest NOX-G15 as a therapeutic candidate with the potential to acutely attenuate hyperglycemia in type 1 and type 2 diabetes.