RESUMEN
Stereoanomalous (SA) subjects have normal visual acuity but reduced stereopsis and may have a prevalence of up to 30%. It has been suggested that, in SA subjects, an imbalance in interocular inhibition might underlie an asymmetry in sensory eye dominance (SED). Our study expands upon previous findings by examining binocular rivalry (BR) mean dominance durations, dichoptic masking (DM) thresholds and SED for a group of SA subjects compared to naïve controls. We examined BR dominance durations and DM thresholds for 15 stereonormal (SN) subjects and 10 SA subjects with normal or corrected-to-normal visual acuity. All subjects had visual acuity of 20/40 or better and less than or equal to two lines difference between eyes. Individuals who scored ≥6/9 on the Randot stereo test and <100 arcmin on the PacMan Stereo Acuity test were considered SN. We compared near-vertical and near-horizontal oriented sine-wave gratings for BR and DM in order to dissociate stereo-related mechanisms that rely on horizontal disparities from other eye-based integration mechanisms. Mean randot scores for SN subjects were 8.5/9 with a PacMan stereoacuity of 33 arcmin, and SA subjects scored 2.5/9 and 3,380 arcmin, respectively. The mean difference in SED was 0.19 for SN and 0.48 for SA when measured with a neutral density filter bar. The SA group showed a large interocular difference in BR durations that was significantly greater than normal (p = 0.004) and correlated with loss of stereoacuity. Moreover, the interocular difference for DM was similarly greater for SA subjects (p = 0.04) although a proportional difference in monocular sensitivity could partially account for this. We also found that both SN and SA subjects presented higher DM thresholds and, to some extent, sensitivity for vertical than horizontal orientations. SA subjects show an abnormal bias toward their dominant eye for both BR and DM. These data suggest that common mechanisms of monocular sensitivity and interocular inhibition may limit multiple binocular measures and provides a practical link to better understand the heterogeneity of stereopsis in amblyopia.
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Percepción de Profundidad/fisiología , Predominio Ocular/fisiología , Trastornos de la Percepción/fisiopatología , Agudeza Visual/fisiología , Adulto , Femenino , Humanos , Masculino , Enmascaramiento Perceptual/fisiología , Visión Binocular/fisiología , Adulto JovenRESUMEN
A hallmark of type 2 diabetes is impaired insulin receptor (IR) signaling that results in dysregulation of glucose homeostasis. Understanding the molecular origins and progression of diabetes and developing therapeutics depend on experimental models of hyperglycemia, hyperinsulinemia, and insulin resistance. We present a novel monoclonal antibody, IRAB-B, that is a specific, potent IR antagonist that creates rapid and long-lasting insulin resistance. IRAB-B binds to the IR with nanomolar affinity and in the presence of insulin efficiently blocks receptor phosphorylation within minutes and is sustained for at least 3 days in vitro. We further confirm that IRAB-B antagonizes downstream signaling and metabolic function. In mice, a single dose of IRAB-B induces rapid onset of hyperglycemia within 6 h, and severe hyperglycemia persists for 2 weeks. IRAB-B hyperglycemia is normalized in mice treated with exendin-4, suggesting that this model can be effectively treated with a GLP-1 receptor agonist. Finally, a comparison of IRAB-B with the IR antagonist S961 shows distinct antagonism in vitro and in vivo. IRAB-B appears to be a powerful tool to generate both acute and chronic insulin resistance in mammalian models to elucidate diabetic pathogenesis and evaluate therapeutics.
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Anticuerpos Monoclonales/uso terapéutico , Resistencia a la Insulina/fisiología , Receptor de Insulina/metabolismo , Animales , Western Blotting , Línea Celular , Diabetes Mellitus Tipo 2 , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/antagonistas & inhibidores , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Humanos , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/metabolismo , Ratones , Ratones Endogámicos C57BL , Péptidos/farmacología , Fosforilación , Unión Proteica , Receptor de Insulina/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND AND PURPOSE: Asthma presents as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyper-reactivity (AHR). Spleen tyrosine kinase (Syk) mediates allergen-induced mast cell degranulation, a central component of allergen-induced inflammation and AHR. However, the role of Syk in IgE-mediated constriction of human small airways remains unknown. In this study, we addressed whether selective inhibition of Syk attenuates IgE-mediated constriction and mast cell mediator release in human small airways. EXPERIMENTAL APPROACH: Human precision cut lung slices (hPCLS) ex vivo derived from non-asthmatic donors were incubated overnight with human IgE, dexamethasone, montelukast, antihistamines or a selective Syk inhibitor (SYKi). High-affinity IgE receptor (FcεRI) activation by anti-IgE cross-linking was performed, and constriction and mediator release measured. Airway constriction was normalized to that induced by maximal carbachol stimulation. Syk expression (determined by qPCR and immunoblot) was also evaluated in human primary airway smooth muscle (HASM) cells to determine whether Syk directly modulates HASM function. KEY RESULTS: While dexamethasone had little effect on FcεR-mediated contraction, montelukast or antihistamines partially attenuated the response. SYKi abolished anti-IgE-mediated contraction and suppressed the release of mast cell or basophil mediators from the IgE-treated hPCLS. In contrast, SYKi had little effect on the non-allergic contraction induced by carbachol. Syk mRNA and protein were undetectable in HASM cells. CONCLUSIONS AND IMPLICATIONS: A selective Syk inhibitor, but not corticosteroids, abolished FcεR-mediated contraction in human small airways ex vivo. The mechanism involved FcεRI receptor activation on mast cells or basophils that degranulate causing airway constriction, rather than direct actions on HASM.
Asunto(s)
Inmunoglobulina E/inmunología , Pulmón/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Bazo/enzimología , Células Cultivadas , Humanos , Técnicas In Vitro , Pulmón/citología , Pulmón/enzimología , Pulmón/inmunología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/inmunología , Músculo Liso/enzimología , Músculo Liso/inmunología , Inhibidores de Proteínas Quinasas/química , Proteínas Tirosina Quinasas/metabolismoRESUMEN
Although much speculation has surrounded intestinally expressed FcRn as a means for systemic uptake of orally administered immunoglobulin G (IgG), this has not been validated in translational models beyond neonates or in FcRn-expressing cells in vitro. Recently, IgG1 intestinal infusion acutely in anesthetized cynomolgus resulted in detectable serum monoclonal antibody (mAb) levels. In this study, we show that IgG2 has greater protease resistance to intestinal enzymes in vitro and mice in vivo, due to protease resistance in the hinge region. An IgG2 mAb engineered for FcRn binding, was optimally formulated, lyophilized, and loaded into enteric-coated capsules for oral dosing in cynomolgus. Small intestinal pH 7.5 was selected for enteric delivery based on gastrointestinal pH profiling of cynomolgus by operator-assisted IntelliCap System(®). Milling of the lyophilized IgG2 M428L FcRn-binding variant after formulation in 10 mmol/L histidine, pH 5.7, 8.5% sucrose, 0.04% PS80 did not alter the physicochemical properties nor the molecular integrity compared to the batch released in PBS. Size 3 hard gel capsules (23.2 mg IgG2 M428L ~3 mg/kg) were coated with hydroxypropyl methylcellulose acetate succinate for rapid dissolution at pH 7.5 in small intestine and FcRn binding of encapsulated mAb confirmed. Initial capsule dosing by endoscopic delivery into the small intestine achieved 0.2 + 0.1 ng/mL (n = 5) peak at 24 h. Weekly oral capsule dosing for 6 weeks achieved levels of 0.4 + 0.2 ng/mL and, despite increasing the dose and frequency, remained below 1 ng/mL. In conclusion, lyophilized milled mAb retains FcRn binding and molecular integrity for small intestinal delivery. The low systemic exposure has demonstrated the limitations of intestinal FcRn in non-human primates and the unfeasibility of employing this for therapeutic levels of mAb. Local mAb delivery with limited systemic exposure may be sufficient as a therapeutic for intestinal diseases.
RESUMEN
Insulin receptor signaling is a complex cascade leading to a multitude of intracellular functional responses. Three natural ligands, insulin, IGF1 and IGF2, are each capable of binding with different affinities to the insulin receptor, and result in variable biological responses. However, it is likely these affinity differences alone cannot completely explain the myriad of diverse cellular outcomes. Ligand binding initiates activation of a signaling cascade resulting in phosphorylation of the IR itself and other intracellular proteins. The direct catalytic activity along with the temporally coordinated assembly of signaling proteins is critical for insulin receptor signaling. We hypothesized that determining differential phosphorylation among individual tyrosine sites activated by ligand binding or dephosphorylation by phosphatases could provide valuable insight into insulin receptor signaling. Here, we present a sensitive, novel immunoassay adapted from Meso Scale Discovery technology to quantitatively measure changes in site-specific phosphorylation levels on endogenous insulin receptors from HuH7 cells. We identified insulin receptor phosphorylation patterns generated upon differential ligand activation and phosphatase-mediated deactivation. The data demonstrate that insulin, IGF1 and IGF2 elicit different insulin receptor phosphorylation kinetics and potencies that translate to downstream signaling. Furthermore, we show that insulin receptor deactivation, regulated by tyrosine phosphatases, occurs distinctively across specific tyrosine residues. In summary, we present a novel, quantitative and high-throughput assay that has uncovered differential ligand activation and site-specific deactivation of the insulin receptor. These results may help elucidate some of the insulin signaling mechanisms, discriminate ligand activity and contribute to a better understanding of insulin receptor signaling. We propose this methodology as a powerful approach to characterize agonists and antagonists of the insulin receptor and can be adapted to serve as a platform to evaluate ligands of alternate receptor systems.
Asunto(s)
Fosfoserina/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptor de Insulina/metabolismo , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Humanos , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Factor II del Crecimiento Similar a la Insulina/farmacología , Cinética , Ligandos , Modelos Biológicos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Tiempo , Vanadatos/farmacologíaRESUMEN
The neonatal Fc receptor (FcRn) in intestinal epithelium is the primary mechanism for transfer of maternal immunoglobulin G (IgG) from suckled milk to serum; but the factors contributing to the rapid uptake of IgG are poorly understood. These studies help to determine the contribution of cell surface FcRn in IgG uptake in 2-week-old rat pups by varying local pH and binding conditions. Variants of a human wild-type (WT) IgG monoclonal antibody (mAb WT) were assessed for binding affinity (KD) to rat (r)FcRn at pH 6.0 and subsequent off-rate at pH 7.4 (1/s) by surface plasmon resonance. Selected mAbs were administered intra-intestinally in isoflurane-anesthetized 2-week rat pups. Full length mAb in serum was quantified by immunoassay, (r)FcRn mRNA expression by reverse transcription polymerase chain reaction, and mAb epithelial localization was visualized by immunohistochemistry. After duodenal administration, serum levels of mAb variants correlated with their rFcRn off-rate at pH 7.4, but not their affinity at pH 6.0. The greatest serum levels of IgG were measured when mAb was administered in the duodenum where rFcRn mRNA expression is greatest, and was increased further by duodenal administration in pH 6.0 buffer. More intense human IgG immunostaining was detected in epithelium than the same variant administered at higher pH. These data suggest an increased contribution for cell surface receptor. We conclude that, in the neonate duodenum, receptor off-rates are as important as affinities for FcRn mediated uptake, and cell surface binding of IgG to rFcRn plays contributes to IgG uptake alongside pinocytosis; both of which responsible for increased IgG uptake.
RESUMEN
PURPOSE: To evaluate transcytosis of immunoglobulin G (IgG) by the neonatal Fc receptor (FcRn) in adult primate intestine to determine whether this is a means for oral delivery of monoclonal antibodies (mAbs). METHODS: Relative regional expression of FcRn and localization in human intestinal mucosa by RT-PCR, ELISA & immunohistochemistry. Transcytosis of full-length mAbs (sandwich ELISA-based detection) across human intestinal segments mounted in Ussing-type chambers, human intestinal (caco-2) cell monolayers grown in transwells, and serum levels after regional intestinal delivery in isoflurane-anesthetized cynomolgus monkeys. RESULTS: In human intestine, there was an increasing proximal-distal gradient of mucosal FcRn mRNA and protein expression. In cynomolgus, serum mAb levels were greater after ileum-proximal colon infusion than after administration to stomach or proximal small intestine (1-5 mg/kg). Serum levels of wild-type mAb dosed into ileum/proximal colon (2 mg/kg) were 124 ± 104 ng/ml (n = 3) compared to 48 ± 48 ng/ml (n = 2) after a non-FcRn binding variant. In vitro, mAb transcytosis in polarized caco-2 cell monolayers and was not enhanced by increased apical cell surface IgG binding to FcRn. An unexpected finding in primate small intestine, was intense FcRn expression in enteroendocrine cells (chromagranin A, GLP-1 and GLP-2 containing). CONCLUSIONS: In adult primates, FcRn is expressed more highly in distal intestinal epithelial cells. However, mAb delivery to that region results in low serum levels, in part because apical surface FcRn binding does not influence mAb transcytosis. High FcRn expression in enteroendocrine cells could provide a novel means to target mAbs for metabolic diseases after systemic administration.
Asunto(s)
Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase I/biosíntesis , Inmunoglobulina G/metabolismo , Mucosa Intestinal/metabolismo , Receptores Fc/biosíntesis , Transcitosis/fisiología , Adulto , Animales , Células CACO-2 , Femenino , Humanos , Macaca fascicularis , Masculino , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , ARN Mensajero/biosíntesis , Ratas , Adulto JovenRESUMEN
Monoclonal antibody (mAb) engineering that optimizes binding to receptors present on brain vascular endothelial cells has enabled them to cross through the blood-brain barrier (BBB) and access the brain parenchyma to treat neurological diseases. However, once in the brain the extent to which receptor-mediated reverse transcytosis clears mAb from the brain is unknown. The aim of this study was to determine the contribution of the neonatal Fc-receptor (FcRn) in rat brain efflux employing two different in vivo drug delivery models. Two mAb variants with substantially different affinities to FcRn, and no known neuronal targets, (IgG1 N434A and H435A) were administered to rats via intranasal-to-central nervous system (CNS) and intra-cranial dosing techniques. Levels of full-length IgG were quantified in serum and brain hemispheres by a sensitive enzyme-linked immunosorbent assay (ELISA). Following intra-nasal delivery, low cerebral hemisphere levels of variants were obtained at 20min, with a trend towards faster clearance of the high FcRn binder (N434A); however, the relatively higher serum levels confounded analysis of brain FcRn contribution to efflux. Using stereotaxic coordinates, we optimized the timing and dosing regimen for injection of mAb into the cortex. Levels of N434A, but not H435A, decreased in the cerebral hemispheres following bilateral injection into the rat cortex and higher levels of N434A were detected in serum compared to H435A after 24h. Immunohistochemical staining of human IgG1 in sections of cortex was consistent with these results, illustrating relatively less intense immunostaining in N434A than H435A dosed animals. Using two in vivo methods with direct cranial administration, we conclude that FcRn plays an important role in efflux of IgG from the rat brain.
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Anticuerpos Monoclonales/farmacocinética , Encéfalo/inmunología , Encéfalo/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulina G/metabolismo , Receptores Fc/inmunología , Animales , Anticuerpos Monoclonales/sangre , Sistemas de Liberación de Medicamentos , Inmunoglobulina G/sangre , Masculino , Mucosa Olfatoria/inmunología , Ratas , Ratas Sprague-DawleyRESUMEN
The pharmacological and airways relaxant profiles of PL-3994 (Hept-cyclo(Cys-His-Phe-d-Ala-Gly-Arg-d-Nle-Asp-Arg-Ile-Ser-Cys)-Tyr-[Arg mimetic]-NH(2)), a novel natriuretic peptide receptor-A (NPR-A) agonist, were evaluated. PL-3994, a full agonist, has high affinity for recombinant human (h), dog, or rat NPR-As (K(i)s of 1, 41, and 10 nm, respectively), and produced concentration-dependent cGMP generation in human, dog and rat NPR-As (respective EC(50)s of 2, 3 and 14 nm). PL-3994 has a K(i) of 7 nm for hNPR-C but was without effect on cGMP generation in hNPR-B. PL-3994 (1 µm) was without significant effect against 75 diverse molecular targets. PL-3994 or BNP, a natural NPR ligand, produced concentration-dependent relaxation of pre-contracted guinea-pig trachea (IC(50)s of 42.7 and 10.7 nm, respectively). PL-3994, and also BNP, (0.1 nm-100 µm) elicited a potent, concentration-dependent but small relaxation of pre-contracted human precision-cut lung slices (hPCLS). Intratracheal PL-3994 (1-1000 µg/kg) produced a dose-dependent inhibition of the bronchoconstrictor response evoked by aerosolized methacholine, but was without significant effect on cardiovascular parameters. PL-3994 was resistant to degradation by human neutral endopeptidase (hNEP) (92% remaining after 2 h), whereas the natural ligands, ANP and CNP, were rapidly metabolized (≤1% remaining after 2 h). PL-3994 is a potent, selective NPR agonist, resistant to NEP, with relaxant effects in guinea-pig and human airway smooth muscle systems. PL-3994 has the profile predictive of longer clinical bronchodilator activity than observed previously with ANP, and suggests its potential utility in the treatment of asthma, in addition to being a useful research tool to evaluate NPR biology.
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Broncodilatadores/farmacología , Neprilisina/metabolismo , Péptidos Cíclicos/farmacología , Piperazinas/farmacología , Receptores del Factor Natriurético Atrial/agonistas , Animales , Perros , Relación Dosis-Respuesta a Droga , Cobayas , Células HEK293 , Humanos , Masculino , Péptido Natriurético Encefálico/farmacología , Péptidos Cíclicos/metabolismo , Ratas , Ratas Wistar , Tráquea/efectos de los fármacos , Tráquea/fisiologíaRESUMEN
In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through unknown mechanisms characterized by increased lung airway smooth muscle (ASM) mass and stiffness. We explored the role of a Regulator of G-protein Signaling protein (RGS4) in the ASM hyperplasia and reduced contractile capacity characteristic of advanced asthma. Using immunocytochemical staining, ASM expression of RGS4 was determined in endobronchial biopsies from healthy subjects and those from subjects with mild, moderate and severe asthma. Cell proliferation assays, agonist-induced calcium mobilization and bronchoconstriction were determined in cultured human ASM cells and in human precision cut lung slices. Using gain- and loss-of-function approaches, the precise role of RGS proteins was determined in stimulating human ASM proliferation and inhibiting bronchoconstriction. RGS4 expression was restricted to a subpopulation of ASM and was specifically upregulated by mitogens, which induced a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. RGS4 expression was markedly increased in bronchial smooth muscle of patients with severe asthma, and expression correlated significantly with reduced pulmonary function. Whereas RGS4 inhibited G protein-coupled receptor (GPCR)-mediated bronchoconstriction, unexpectedly RGS4 was required for PDGF-induced proliferation and sustained activation of PI3K, a mitogenic signaling molecule that regulates ASM proliferation. These studies indicate that increased RGS4 expression promotes a phenotypic switch of ASM, evoking irreversible airway obstruction in subjects with severe asthma.
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Obstrucción de las Vías Aéreas/patología , Asma/complicaciones , Asma/patología , Bronquios/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Proteínas RGS/metabolismo , Obstrucción de las Vías Aéreas/complicaciones , Obstrucción de las Vías Aéreas/fisiopatología , Asma/fisiopatología , Broncoconstricción/efectos de los fármacos , Carbacol/farmacología , Proliferación Celular/efectos de los fármacos , Acoplamiento Excitación-Contracción/efectos de los fármacos , Femenino , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Mitógenos/farmacología , Modelos Biológicos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/enzimología , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esputo/efectos de los fármacos , Esputo/metabolismoRESUMEN
Severe asthma is associated with fixed airway obstruction attributable to inflammation, copious luminal mucus, and increased airway smooth muscle (ASM) mass. Paradoxically, studies demonstrated that the hypertrophic and hyperplastic ASM characteristic of severe asthma has reduced contractile capacity. We compared the G-protein-coupled receptor (GPCR)-induced Ca(2+) mobilization and expression of GPCRs and signaling proteins related to procontractile signaling in ASM derived postmortem from subjects who died of nonrespiratory causes, with cells from subjects who died of asthma. Despite the increased or comparable expression of contraction-promoting GPCRs (bradykinin B2 or histamine H1 and protease-activated receptor 1, respectively) in asthmatic ASM cells relative to cells from healthy donors, asthmatic ASM cells exhibited reduced histamine-induced Ca(2+) mobilization and comparable responses to bradykinin and thrombin, suggesting a postreceptor signaling defect. Accordingly, the expression of regulator of G-protein signaling-5 (RGS5), an inhibitor of ASM contraction, was increased in cultured, asthmatic ASM cells and in bronchial smooth muscle bundles of both human subjects with asthma and allergen-challenged mice, relative to those of healthy human subjects or naive mice. The overexpression of RGS5 impaired the release of Ca(2+) to thrombin, histamine, and carbachol, and reduced the contraction of precision-cut lung slices to carbachol. These results suggest that increased RGS5 expression contributes to decreased myocyte shortening in severe and fatal asthma.
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Asma/metabolismo , Bronquios/metabolismo , Proteínas de Unión al GTP/metabolismo , Transducción de Señal , Adulto , Asma/patología , Bronquios/patología , Femenino , Humanos , Persona de Mediana Edad , Contracción MuscularRESUMEN
Atopic asthma is poorly controlled by current therapies. Newer therapies and novel antihistamines are, therefore, required to treat patients whose atopic asthma is not controlled. For the first time, C-027 is shown to antagonize histamine, IgE-mediated and serotonin-induced contraction in human airways and vessels. Human precision-cut lung slices (PCLS, 250 µm thick), containing an airway or blood vessel, were pretreated with either C-027 (2 hours) or with vehicle alone and were contracted with histamine or serotonin. Known antihistamine was used as a comparator in antihistamine studies. Also, human airways were contracted via IgE passive sensitization in the presence or absence of C-027 or fexofenadine. Affinity of C-027 toward human G-protein coupled receptors was also determined, as well as the drug's biodistribution in murine model. C-027 was shown to have the highest affinity toward human histamine and serotonin receptors. Subsequently, C-027 was shown to antagonize histamine- and serotonin-induced airway and vascular smooth muscle contraction, respectively, and histamine-released bronchocontraction mediated by IgE passive sensitization in human small airways. C-027 also inhibited histamine-mediated single-cell calcium ion release. Low levels of C-027 were found in murine brain tissue. Collectively, these data suggest that C-027 markedly inhibits IgE-induced bronchoconstriction and antagonizes histamine and serotonin-contraction with little biodistribution in the brain. The compound may offer a future therapy for allergen-induced airway hyperresponsiveness in patients with asthma.
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Acetamidas/farmacología , Broncoconstricción/efectos de los fármacos , Broncoconstricción/inmunología , Antagonistas de los Receptores Histamínicos/farmacología , Inmunoglobulina E/inmunología , Fenotiazinas/farmacología , Antagonistas de la Serotonina/farmacología , Acetamidas/metabolismo , Animales , Disponibilidad Biológica , Hiperreactividad Bronquial/inmunología , Calcio/metabolismo , Histamina/farmacología , Antagonistas de los Receptores Histamínicos/metabolismo , Humanos , Hipersensibilidad Inmediata/inmunología , Ratones , Fenotiazinas/metabolismo , Piperazinas , Receptores Histamínicos/metabolismo , Sistema Respiratorio/efectos de los fármacos , Sistema Respiratorio/inmunología , Antagonistas de la Serotonina/metabolismoRESUMEN
Although short-acting and long-acting inhaled ß(2)-adrenergic receptor agonists (SABA and LABA, respectively) relieve asthma symptoms, use of either agent alone without concomitant anti-inflammatory drugs (corticosteroids) may increase the risk of disease exacerbation in some patients. We found previously that pretreatment of human precision-cut lung slices (PCLS) with SABA impaired subsequent ß(2)-agonist-induced bronchodilation, which occurred independently of changes in receptor quantities. Here we provide evidence that prolonged exposure of cultured human airway smooth muscle (HuASM) cells to ß(2)-agonists directly augments procontractile signaling pathways elicited by several compounds including thrombin, bradykinin, and histamine. Such treatment did not increase surface receptor amounts or expression of G proteins and downstream effectors (phospholipase Cß and myosin light chain). In contrast, ß-agonists decreased expression of regulator of G protein signaling 5 (RGS5), which is an inhibitor of G-protein-coupled receptor (GPCR) activity. RGS5 knockdown in HuASM increased agonist-evoked intracellular calcium flux and myosin light chain (MLC) phosphorylation, which are prerequisites for contraction. PCLS from Rgs5(-/-) mice contracted more to carbachol than those from WT mice, indicating that RGS5 negatively regulates bronchial smooth muscle contraction. Repetitive ß(2)-agonist use may not only lead to reduced bronchoprotection but also to sensitization of excitation-contraction signaling pathways as a result of reduced RGS5 expression.
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Agonistas de Receptores Adrenérgicos beta 2/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Pulmón/metabolismo , Músculo Liso/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas RGS/biosíntesis , Transducción de Señal/efectos de los fármacos , Animales , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Contracción Muscular/efectos de los fármacos , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Fosforilación/efectos de los fármacos , Proteínas RGS/genética , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: During pregnancy asthma may remain stable, improve or worsen. The factors underlying the deleterious effect of pregnancy on asthma remain unknown. Oxytocin is a neurohypophyseal protein that regulates a number of central and peripheral responses such as uterine contractions and milk ejection. Additional evidence suggests that oxytocin regulates inflammatory processes in other tissues given the ubiquitous expression of the oxytocin receptor. The purpose of this study was to define the role of oxytocin in modulating human airway smooth muscle (HASMCs) function in the presence and absence of IL-13 and TNFalpha, cytokines known to be important in asthma. METHOD: Expression of oxytocin receptor in cultured HASMCs was performed by real time PCR and flow cytomery assays. Responses to oxytocin was assessed by fluorimetry to detect calcium signals while isolated tracheal rings and precision cut lung slices (PCLS) were used to measure contractile responses. Finally, ELISA was used to compare oxytocin levels in the bronchoalveloar lavage (BAL) samples from healthy subjects and those with asthma. RESULTS: PCR analysis demonstrates that OXTR is expressed in HASMCs under basal conditions and that both interleukin (IL)-13 and tumor necrosis factor (TNFalpha) stimulate a time-dependent increase in OXTR expression at 6 and 18 hr. Additionally, oxytocin increases cytosolic calcium levels in fura-2-loaded HASMCs that were enhanced in cells treated for 24 hr with IL-13. Interestingly, TNFalpha had little effect on oxytocin-induced calcium response despite increasing receptor expression. Using isolated murine tracheal rings and PCLS, oxytocin also promoted force generation and airway narrowing. Further, oxytocin levels are detectable in bronchoalveolar lavage (BAL) fluid derived from healthy subjects as well as from those with asthma. CONCLUSION: Taken together, we show that cytokines modulate the expression of functional oxytocin receptors in HASMCs suggesting a potential role for inflammation-induced changes in oxytocin receptor signaling in the regulation of airway hyper-responsiveness in asthma.
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Asma/metabolismo , Interleucina-13/metabolismo , Pulmón/metabolismo , Miocitos del Músculo Liso/metabolismo , Oxitocina/metabolismo , Receptores de Oxitocina/metabolismo , Tráquea/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Animales , Asma/inmunología , Asma/fisiopatología , Líquido del Lavado Bronquioalveolar/química , Broncoconstricción , Broncodilatadores/farmacología , Señalización del Calcio , Estudios de Casos y Controles , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Fluorometría , Volumen Espiratorio Forzado , Humanos , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/fisiopatología , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/inmunología , ARN Mensajero/metabolismo , Receptores de Oxitocina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Tráquea/efectos de los fármacos , Tráquea/inmunología , Tráquea/fisiopatología , Regulación hacia Arriba , Capacidad Vital , Adulto JovenRESUMEN
BACKGROUND: Ozone, a pollutant known to induce airway hyper-responsiveness (AHR), increases morbidity and mortality in patients with obstructive airway diseases and asthma. We postulate oxidized lipids mediate in vivo ozone-induced AHR in murine airways. METHODOLOGY/PRINCIPAL FINDINGS: Male BALB/c mice were exposed to ozone (3 or 6 ppm) or filtered air (controls) for 2 h. Precision cut lung slices (PCLS; 250 microm thickness) containing an intrapulmonary airway ( approximately 0.01 mm(2) lumen area) were prepared immediately after exposure or 16 h later. After 24 h, airways were contracted to carbachol (CCh). Log EC(50) and E(max) values were then calculated by measuring the airway lumen area with respect to baseline. In parallel studies, dexamethasone (2.5 mg/kg), or 1-aminobenzotriazol (ABT) (50 mg/kg) were given intraperitoneal injection to naïve mice 18 h prior to ozone exposure. Indomethacin (10 mg/kg) was administered 2 h prior. Cell counts, cytokine levels and liquid chromatography-mass spectrometry (LC-MS) for lipid analysis were assessed in bronchoalveolar lavage (BAL) fluid from ozone exposed and control mice. Ozone acutely induced AHR to CCh. Dexamethasone or indomethacin had little effect on the ozone-induced AHR; while, ABT, a cytochrome P450 inhibitor, markedly attenuated airway sensitivity. BAL fluid from ozone exposed animals, which did not contain an increase in neutrophils or interleukin (IL)-6 levels, increased airway sensitivity following in vitro incubation with a naïve PCLS. In parallel, significant increases in oxidized lipids were also identified using LC-MS with increases of 20-HETE that were decreased following ABT treatment. CONCLUSIONS/SIGNIFICANCE: These data show that ozone acutely induces AHR to CCh independent of inflammation and is insensitive to steroid treatment or cyclooxygenase (COX) inhibition. BAL fluid from ozone exposed mice mimicked the effects of in vivo ozone exposure that were associated with marked increases in oxidized lipids. 20-HETE plays a pivotal role in mediating acute ozone-induced AHR.
Asunto(s)
Ácidos Hidroxieicosatetraenoicos/análisis , Ácidos Hidroxieicosatetraenoicos/inmunología , Ozono/farmacología , Hipersensibilidad Respiratoria/etiología , Animales , Hiperreactividad Bronquial , Carbacol/farmacología , Peroxidación de Lípido , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB C , Neutrófilos , Triazoles/farmacologíaRESUMEN
Nonsteroidal anti-inflammatory drugs ameliorate pain and fever by inhibiting cyclooxygenase (COX) and suppressing prostanoid formation. Microsomal prostaglandin E synthase-1 (mPGES-1) catalyzes formation of PGE(2) from the COX product PGH(2) and has emerged as a therapeutic target. Inhibition of mPGES-1, however, renders the PGH(2) substrate available for diversion to other PG synthases. To address the possibility that substrate diversion augments formation of PGs that might modulate bronchial tone, we assessed the impact of mPGES-1 deletion in a mouse model of ozone-induced airway hyper-responsiveness. Ozone exposure increased total lung resistance to inhaled methacholine in wild-type mice. Deletion of mPGES-1 had little effect on total lung resistance in either naive or ozone-exposed animals. The carbachol-induced narrowing of luminal diameter in intrapulmonary airways of lung slices from acute ozone-exposed mice was also unaltered by mPGES-1 deletion. Likewise, although concentrations of PGE(2) were reduced in bronchoalveolar lavage fluid, whereas 6-keto-PGF(1alpha), PGD(2), and PGF(2alpha), all were increased, deletion of mPGES-1 failed to influence cell trafficking into the airways of either naive or ozone-exposed animals. Despite biochemical evidence of PGH(2) substrate diversion to potential bronchomodulator PGs, deletion of mPGES-1 had little effect on ozone-induced airway inflammation or airway hyper-responsiveness. Pharmacologically targeting mPGES-1 may not predispose patients at risk to airway dysfunction.
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Resistencia de las Vías Respiratorias/efectos de los fármacos , Dinoprostona/biosíntesis , Eliminación de Gen , Oxidorreductasas Intramoleculares/fisiología , Ozono/farmacología , Sistema Respiratorio/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/efectos adversos , Carbacol/farmacología , Diferenciación Celular/efectos de los fármacos , Femenino , Oxidorreductasas Intramoleculares/deficiencia , Cloruro de Metacolina/farmacología , Ratones , Ratones Noqueados , Microsomas/enzimología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Prostaglandina-E Sintasas , Prostaglandinas/biosíntesis , Sistema Respiratorio/enzimología , Sistema Respiratorio/inmunología , Sistema Respiratorio/metabolismoRESUMEN
Chronic obstructive pulmonary disease (COPD) involves disease of small airways with an increase in airway smooth muscle sensitivity to spasmogens and with structural changes described as airway remodeling. We investigated the effect of tobacco smoke (TS) exposure on the structure and function of small airways in rats and the role of IL-13 in this response. Precision-cut lung slices (230-280 microm) were prepared from male Sprague-Dawley rats after acute (3 d) or chronic (8 or 16 wk) daily exposure to TS or air. Carbachol (CCh) and 5-hydroxytryptamine (5HT) concentration responses were performed on airways (50-400 microm diameter). The effect of IL-13 in vitro on small airway sensitivity to CCh and 5HT was also determined. Acute exposure to TS did not affect the sensitivity of the intrapulmonary airways to either spasmogen. After 8 weeks of TS exposure, airway hyperresponsiveness (AHR) to CCh was evident (log EC(50) CCh: air = 0.22 microM; TS = -0.12 microM; P = 0.019); AHR to 5HT was also observed after the 16-week exposure to TS (air = -0.85 microM; TS = -1.06 microM; P = 0.038). Chronic TS exposure increased airway wall SMA content, which correlated with increased expression of IL-13 and transforming growth factor (TGF)-beta(1) in the lung tissues. In vitro incubation with IL-13, but not TGF-beta(1), induced changes in small airway sensitivity to CCh and 5HT. Chronic TS exposure induces increased responsiveness in intrapulmonary airways of rats that may be mediated in part by an increase in IL-13.
Asunto(s)
Interleucina-13/fisiología , Pulmón/metabolismo , Nicotiana/metabolismo , Actinas/metabolismo , Animales , Bronquios/efectos de los fármacos , Bronquios/patología , Hiperreactividad Bronquial/fisiopatología , Colágeno/química , Modelos Animales de Enfermedad , Interleucina-13/metabolismo , Masculino , Músculo Liso/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Ratas , Ratas Sprague-Dawley , Humo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Respiratory infections exacerbate chronic lung diseases promoting airway inflammation and hyperreactivity. Toll-like receptor 3 (TLR3) recognizes viral double-stranded (ds) RNA such as polyinosinic-polycytidylic acid [poly(I:C)] and stimulates innate immune responses. The objective of this study was to test the hypothesis that dsRNA promotes lung inflammation and alters airway responsiveness to cholinergic and beta-adrenergic receptor agonists in human lung slices. Human airway smooth muscle (ASM) was incubated for 24 h in poly(I:C) +/- TNFalpha and a TLR3 monoclonal antibody. Precision-cut lung slices (PCLS; 250-microm thickness) from healthy human lungs containing a small airway were incubated in 0, 10, or 100 microg/ml poly(I:C) for 24 h. Intravital microscopy of lung slices was used to quantify contractile and relaxation responsiveness to carbachol and isoproterenol, respectively. Supernatants of ASM and PCLS were analyzed for cytokine secretion using a 25-multiplex bead assay. In human ASM, poly(I:C) (0.5 microg/ml) increased macrophage inflammatory protein-1alpha (MIP-1alpha) and RANTES that was prevented by a TLR3 monoclonal receptor antibody. Incubation of human PCLS with poly(I:C) (10 and 100 microg/ml) had little effect on the log EC(50) or maximum drug effect (E(max)) for contraction and relaxation in response to carbachol and isoproterenol, respectively. The responsiveness of the same human PCLS to poly(I:C) incubation was confirmed by the robust increase in chemokines and cytokines. In separate experiments, incubation of PCLS with IL-13 or TNFalpha (100 ng/ml) increased airway sensitivity to carbachol. Poly(I:C) promotes inflammatory mediator release that was not associated with enhanced bronchoconstriction or attenuated bronchodilation in normal healthy human lung slices. Transduction at the TLR3 initiated by dsRNA stimulates downstream innate immune responses.
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Citocinas/metabolismo , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Poli I-C/farmacología , Receptor Toll-Like 3/metabolismo , Tráquea/efectos de los fármacos , Tráquea/fisiología , Quimiocinas/metabolismo , Humanos , Técnicas In Vitro , Interleucina-13/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Tráquea/citología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND: Evidence suggests that chronic stimulation of beta(2)-adrenergic receptors (beta(2)-ARs) induces receptor tolerance that limits the efficacy of beta-agonists in the treatment of asthma. The precise mechanisms that induce beta(2)-AR tolerance remain unclear. OBJECTIVE: We sought to determine whether steroids modulate albuterol-induced beta(2)-AR tolerance in human small airways. METHODS: beta(2)-AR responsiveness to isoproterenol was characterized in human precision-cut lung slices (PCLSs) precontracted to carbachol after pretreatment with albuterol. RESULTS: Incubation of PCLSs with albuterol for 3, 6, or 12 hours attenuated subsequent isoproterenol-induced relaxation in a dose- and time-dependent manner. A 40% decrease (P < .0001) in maximum relaxation and a 45% decrease (P = .0011) in airway sensitivity from control values occurred after the maximum time and concentration of albuterol incubation. Desensitization was not evident when airways were relaxed to forskolin. Dexamethasone pretreatment of PCLSs (1 hour) prevented albuterol-induced beta(2)-AR desensitization by increasing the maximum drug effect (P = .0023) and decreasing the log half-maximum effective concentration values (P < .0001) from that of albuterol alone. Albuterol (12-hour incubation) decreased the beta(2)-AR cell-surface number (P = .013), which was not significantly reversed by 1 hour of preincubation with dexamethasone. CONCLUSION: These data suggest that beta(2)-AR desensitization occurs with prolonged treatment of human small airways with albuterol through mechanisms upstream of protein kinase A and that steroids prevent or reverse this desensitization. Clarifying the precise molecular mechanisms by which beta(2)-AR tolerance occurs might offer new therapeutic approaches to improve the efficacy of bronchodilators in asthma and chronic obstructive pulmonary disease.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2 , Agonistas Adrenérgicos beta/farmacología , Albuterol/farmacología , Antiinflamatorios/farmacología , Asma/fisiopatología , Dexametasona/farmacología , Resistencia a Medicamentos/efectos de los fármacos , Isoproterenol/farmacología , Pulmón , Asma/tratamiento farmacológico , Asma/metabolismo , Broncoconstricción/efectos de los fármacos , Broncodilatadores/farmacología , Humanos , Pulmón/metabolismo , Pulmón/fisiopatología , Técnicas de Cultivo de Órganos , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Receptores Adrenérgicos beta 2/metabolismo , Factores de TiempoRESUMEN
Chronic obstructive pulmonary disease (COPD) is a smoking-related disease that lacks effective therapies due partly to the poor understanding of disease pathogenesis. The aim of this study was to identify molecular pathways that could be responsible for the damaging consequences of smoking. To do this, we employed Gene Set Enrichment Analysis to analyze differences in global gene expression, which we then related to the pathological changes induced by cigarette smoke (CS). Sprague-Dawley rats were exposed to whole body CS for 1 day and for various periods up to 8 mo. Gene Set Enrichment Analysis of microarray data identified that metabolic processes were most significantly increased early in the response to CS. Gene sets involved in stress response and inflammation were also upregulated. CS exposure increased neutrophil chemokines, cytokines, and proteases (MMP-12) linked to the pathogenesis of COPD. After a transient acute response, the CS-exposed rats developed a distinct molecular signature after 2 wk, which was followed by the chronic phase of the response. During this phase, gene sets related to immunity and defense progressively increased and predominated at the later time points in smoke-exposed rats. Chronic CS inhalation recapitulated many of the phenotypic changes observed in COPD patients including oxidative damage to macrophages, a slowly resolving inflammation, epithelial damage, mucus hypersecretion, airway fibrosis, and emphysema. As such, it appears that metabolic pathways are central to dealing with the stress of CS exposure; however, over time, inflammation and stress response gene sets become the most significantly affected in the chronic response to CS.