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OBJECTIVES: Disease activity control in patients with systemic lupus erythematosus (SLE) with corticosteroid and immunosuppressant withdrawal is a treatment goal. We evaluated whether this could be attained with sequential subcutaneous belimumab (BEL) and one cycle of rituximab (RTX). METHODS: In this phase 3, double-blind BLISS-BELIEVE trial (GSK Study 205646), patients with active SLE initiating subcutaneous BEL 200 mg/week for 52 weeks were randomised to intravenous placebo (BEL/PBO) or intravenous RTX 1000 mg (BEL/RTX) at weeks 4 and 6 while stopping concomitant immunosuppressants/tapering corticosteroids; standard therapy for 104 weeks (BEL/ST; reference arm) was included. PRIMARY ENDPOINT: proportion of patients achieving disease control (SLE Disease Activity Index-2000 (SLEDAI-2K) ≤2; without immunosuppressants; prednisone equivalent ≤5 mg/day) at week 52 with BEL/RTX versus BEL/PBO. Major (alpha-controlled) secondary endpoints: proportion of patients with clinical remission (week 64; clinical SLEDAI-2K=0, without immunosuppressants/corticosteroids); proportion of patients with disease control (week 104). Other assessments: disease control duration, anti-dsDNA antibody, C3/C4 and B cells/B-cell subsets. RESULTS: The modified intention-to-treat population included 263 patients. Overall, 16.7% (12/72) of BEL/PBO and 19.4% (28/144) of BEL/RTX patients achieved disease control (OR (95% CI) 1.27 (0.60 to 2.71); p=0.5342) at week 52. For major secondary endpoints, differences between BEL/RTX and BEL/PBO were not statistically significant. Anti-dsDNA antibodies and most assessed B cells/B-cell subsets were lower with BEL/RTX versus BEL/PBO. Mean disease control duration through 52 weeks was significantly greater with BEL/RTX versus BEL/PBO. CONCLUSIONS: BEL/RTX showed no superiority over BEL/PBO for most endpoints analysed; however, it led to significant improvements in disease activity markers compared with BEL/PBO. Further investigation of combination treatment is warranted. TRIAL REGISTRATION NUMBER: NCT03312907.
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BACKGROUNDPrimary Sjögren's syndrome (pSS) is characterized by B cell hyperactivity and elevated B-lymphocyte stimulator (BLyS). Anti-BLyS treatment (e.g., belimumab) increases peripheral memory B cells; decreases naive, activated, and plasma B cell subsets; and increases stringency on B cell selection during reconstitution. Anti-CD20 therapeutics (e.g., rituximab) bind and deplete CD20-expressing B cells in circulation but are less effective in depleting tissue-resident CD20+ B cells. Combined, these 2 mechanisms may achieve synergistic effects.METHODSThis 68-week, phase II, double-blind study (GSK study 201842) randomized 86 adult patients with active pSS to 1 of 4 arms: placebo, s.c. belimumab, i.v. rituximab, or sequential belimumab + rituximab.RESULTSOverall, 60 patients completed treatment and follow-up until week 68. The incidence of adverse events (AEs) and drug-related AEs was similar across groups. Infections/infestations were the most common AEs, and no serious infections of special interest occurred. Near-complete depletion of minor salivary gland CD20+ B cells and a greater and more sustained depletion of peripheral CD19+ B cells were observed with belimumab + rituximab versus monotherapies. With belimumab + rituximab, reconstitution of peripheral B cells occurred, but it was delayed compared with rituximab. At week 68, mean (± standard error) total EULAR Sjögren's syndrome disease activity index scores decreased from 11.0 (1.17) at baseline to 5.0 (1.27) for belimumab + rituximab and 10.4 (1.36) to 8.6 (1.57) for placebo.CONCLUSIONThe safety profile of belimumab + rituximab in pSS was consistent with the monotherapies. Belimumab + rituximab induced enhanced salivary gland B cell depletion relative to the monotherapies, potentially leading to improved clinical outcomes.TRIAL REGISTRATIONClinicalTrials.gov NCT02631538.FUNDINGFunding was provided by GSK.
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Síndrome de Sjögren , Humanos , Rituximab/uso terapéutico , Síndrome de Sjögren/tratamiento farmacológicoRESUMEN
Damage from infestations of Lymantria dispar L. in oak-dominated stands and southern pine beetle (Dendroctonus frontalis Zimmermann) in pine-dominated stands have far exceeded impacts of other disturbances in forests of the mid-Atlantic Coastal Plain over the last two decades. We used forest census data collected in undisturbed and insect-impacted stands combined with eddy covariance measurements made pre- and post-disturbance in oak-, mixed and pine-dominated stands to quantify how these infestations altered forest composition, structure and carbon dynamics in the Pinelands National Reserve of southern New Jersey. In oak-dominated stands, multi-year defoliation during L. dispar infestations resulted in > 40% mortality of oak trees and the release of pine saplings and understory vegetation, while tree mortality was minimal in mixed and pine-dominated stands. In pine-dominated stands, southern pine beetle infestations resulted in > 85% mortality of pine trees but had minimal effect on oaks in upland stands or other hardwoods in lowland stands, and only rarely infested pines in hardwood-dominated stands. Because insect-driven disturbances are both delaying and accelerating succession in stands dominated by a single genus but having less effect in mixed-composition stands, long-term disturbance dynamics are favoring the formation and persistence of uneven age oak-pine mixedwood stands. Changes in forest composition may have little impact on forest productivity and evapotranspiration; although seasonal patterns differ, with highest daily rates of net ecosystem production (NEP) during the growing season occurring in an oak-dominated stand and lowest in a pine-dominated stand, integrated annual rates of NEP are similar among oak-, mixed and pine-dominated stands. Our research documents the formation of mixedwood stands as a consequence of insect infestations in the mid-Atlantic region and suggests that managing for mixedwood stands could reduce damage to forest products and provide greater continuity in ecosystem functioning.
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Escarabajos , Pinus , Quercus , Animales , Ecosistema , Bosques , ÁrbolesRESUMEN
Purpose: Two features define the future of glaucoma therapeutics: (1) greatly improved ocular hypotensive efficacy and (2) a delivery method that improves patient convenience and compliance. A highly efficacious and extraordinarily long-acting ocular hypotensive agent PGN 9856-isopropyl ester represents a potential next-generation anti-glaucoma drug. A new periorbital drug delivery route was also investigated. Methods: PGN 9856-isopropyl ester pharmacology was determined by employing human cells, including prostanoid receptor transfectants, and FLIPr or cellular dielectric spectroscopy technology. Intraocular pressure (IOP) was measured in conscious cynomolgus monkeys trained to accept pneumatonometry when under gentle restraint. For periorbital application, the compound was applied radially using a roller-ball device connected to a cylindrical reservoir. Pharmacokinetic data were obtained using LC/MS/MS instrumentation. Results: Single doses of PGN 9856-isopropyl ester, administered over a 0.001%-0.01% dose range, produced profound decreases in monkey IOP that persisted for at least 5 days, which was long after the drug was detectable in ocular tissues. It was not uncommon for a single eye drop to reduce IOP to the level of 4-7 mm Hg. Drug application to the periorbital dermis of ocular normotensive monkeys produced a similarly profound reduction in IOP, which was well maintained. Conclusions: PGN 9856-isopropyl ester appears to possess efficacy and duration of action properties unmatched by currently prescribed anti-glaucoma agents and by those currently undergoing clinical evaluation. In addition, application to the periorbital skin using a roller-ball device offers a more convenient method of ophthalmic drug delivery than eye drops and is noninvasive, unlike other "dropless" technologies.
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Acetatos/uso terapéutico , Compuestos de Bifenilo/uso terapéutico , Sistemas de Liberación de Medicamentos , Ésteres/uso terapéutico , Glaucoma/tratamiento farmacológico , Soluciones Oftálmicas/uso terapéutico , Acetatos/administración & dosificación , Acetatos/química , Administración Tópica , Animales , Humor Acuoso/química , Compuestos de Bifenilo/administración & dosificación , Compuestos de Bifenilo/química , Córnea/química , Ésteres/administración & dosificación , Ésteres/química , Femenino , Humanos , Macaca fascicularis , Masculino , Soluciones Oftálmicas/administración & dosificación , Soluciones Oftálmicas/química , Imagen Óptica , Factores de TiempoRESUMEN
Activities of ecosystem engineers can interact with other disturbances to modulate rates of key processes such as productivity and nutrient cycling. Bioturbation, movement of soil by organisms, is a widespread form of ecosystem engineering in terrestrial ecosystems. We propose that bioturbation by southeastern pocket gophers (Geomys pinetis), an abundant but declining ecosystem engineer in longleaf pine (Pinus palustris Mill.) forests, accelerates nutrient dynamics of the forest floor by burying litter and then reduces litter consumption and nitrogen (N) volatilization losses in the presence of fire. We evaluated our hypothesis by measuring how litter burial alters decomposition and N and phosphorus (P) turnover of longleaf pine and turkey oak (Quercus laevis Walt.) litter over four years, and then simulated interactive ecosystem-level effects of litter burial and low-intensity fires on N and P dynamics of the litter layer. In the field, mass loss was over two times greater and N and P were released much more rapidly from litter buried beneath mounds than on the surface of the forest floor. At a measured rate of mound formation covering 2.3 ± 0.6% of the forest floor per year, litter mass and N and P content of the forest floor simulated over an eight-year period were approximately 11% less than amounts in areas without pocket gopher mounds. In contrast to unburied litter, litter beneath mounds is protected from consumption during fires, and as fire interval increased, consumption rates decreased because mounds cover more years of accumulated litter. Our research indicates that bioturbation and burial of litter by pocket gophers accelerates turnover of N and P on the forest floor, and in the presence of fire, conserves N in this ecosystem where productivity is known to be nutrient limited.
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Seguimiento de Parámetros Ecológicos/métodos , Ecosistema , Suelo/química , Animales , Incendios , Bosques , Ardillas Terrestres , Mamíferos , Nitrógeno/química , Nutrientes , Valor Nutritivo , Fósforo/química , Pinus , Hojas de la Planta/química , ÁrbolesRESUMEN
A series of potent, selective and long-acting quinoline-based sulfonamide human H1 histamine receptor antagonists, designed for once-daily intranasal administration for the treatment of rhinitis were developed. Sulfonamide 33b had a slightly lower affinity for the H1 receptor than azelastine, had low oral bioavailability in the rat and dog, and was turned over to five major metabolites. Furthermore, 33b had longer duration of action than azelastine in guinea pigs, lower rat brain-penetration, and did not cause time dependent inhibition of CYP2D6 or CYP3A4. The clinical dose in humans is expected to be low (approximately 0.5mg per day) based on the clinical dose used for azelastine and a comparison of efficacy data from animal models for 33b and azelastine.
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Antagonistas de los Receptores Histamínicos H1/química , Quinolinas/química , Receptores Histamínicos H1/metabolismo , Rinitis Alérgica/tratamiento farmacológico , Sulfanilamidas/química , Sulfonamidas/química , Sulfonas/química , Administración Intranasal , Animales , Encéfalo/metabolismo , Perros , Cobayas , Semivida , Antagonistas de los Receptores Histamínicos H1/farmacocinética , Antagonistas de los Receptores Histamínicos H1/uso terapéutico , Concentración 50 Inhibidora , Quinolinas/farmacocinética , Quinolinas/uso terapéutico , Ratas , Receptores Histamínicos H1/química , Rinitis Alérgica/metabolismo , Rinitis Alérgica/patología , Relación Estructura-Actividad , Sulfanilamida , Sulfanilamidas/farmacocinética , Sulfanilamidas/uso terapéutico , Sulfonamidas/farmacocinética , Sulfonamidas/uso terapéutico , Sulfonas/farmacocinética , Sulfonas/uso terapéuticoRESUMEN
The synthesis of potent amide-containing phthalazinone H1 histamine receptor antagonists is described. Three analogues 3e, 3g, and 9g were equipotent with azelastine and were longer-acting in vitro. Amide 3g had low oral bioavailability, low brain-penetration, high metabolic clearance, and long duration of action in vivo, and it was suitable for once-daily dosing intranasally, with a predicted dose for humans of approximately 0.5 mg per day.
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A comparative analysis of the impacts of prescribed fire on three upland forest stands in the Northeastern Atlantic Plain, NJ, USA, was conducted. Effects of prescribed fire on water use and gas exchange of overstory pines were estimated via sap-flux rates and photosynthetic measurements on Pinus rigida Mill. Each study site had two sap-flux plots, one experiencing prescribed fire and one control (unburned) plot for comparison before and after the fire. We found that photosynthetic capacity in terms of Rubisco-limited carboxylation rate and intrinsic water-use efficiency was unaffected, while light compensation point and dark respiration rate were significantly lower in the burned vs control plots post-fire. Furthermore, quantum yield in pines in the pine-dominated stands was less affected than pines in the mixed oak/pine stand, as there was an increase in quantum yield in the oak/pine stand post-fire compared with the control (unburned) plot. We attribute this to an effect of forest type but not fire per se. Average daily sap-flux rates of the pine trees increased compared with control (unburned) plots in pine-dominated stands and decreased in the oak/pine stand compared with control (unburned) plots, potentially due to differences in fuel consumption and pre-fire sap-flux rates. Finally, when reference canopy stomatal conductance was analyzed, pines in the pine-dominated stands were more sensitive to changes in vapor pressure deficit (VPD), while stomatal responses of pines in the oak/pine stand were less affected by VPD. Therefore, prescribed fire affects physiological functioning and water use of pines, but the effects may be modulated by forest stand type and fuel consumption pattern, which suggests that these factors may need to be taken into account for forest management in fire-dominated systems.
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Incendios , Pinus/fisiología , Ecosistema , Bosques , New JerseyRESUMEN
With many safety and technical limitations partly mitigated through chemical modifications, antisense oligonucleotides (ASOs) are gaining recognition as therapeutic entities. The increase in potency realized by 'third generation chemistries' may, however, simultaneously increase affinity to unintended targets with partial sequence complementarity. However, putative hybridization-dependent off-target effects (OTEs), a risk historically regarded as low, are not being adequately investigated. Here we show an unexpectedly high OTEs confirmation rate during screening of fully phosphorothioated (PS)-LNA gapmer ASOs designed against the BACH1 transcript. We demonstrate in vitro mRNA and protein knockdown of off-targets with a wide range of mismatch (MM) and gap patterns. Furthermore, with RNase H1 activity residing within the nucleus, hybridization predicted against intronic regions of pre-mRNAs was tested and confirmed. This dramatically increased ASO-binding landscape together with relatively high potency of such interactions translates into a considerable safety concern. We show here that with base pairing-driven target recognition it is possible to predict the putative off-targets and address the liability during lead design and optimization phases. Moreover, in silico analysis performed against both primary as well as spliced transcripts will be invaluable in elucidating the mechanism behind the hepatoxicity observed with some LNA-modified gapmers.
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Exones , Técnicas de Silenciamiento del Gen , Intrones , Oligonucleótidos Antisentido , Disparidad de Par Base , Células Cultivadas , Simulación por Computador , Silenciador del Gen , Humanos , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/uso terapéutico , Ribonucleasa H/metabolismoRESUMEN
Pine-oak ecosystems are globally distributed even though differences in anatomy and leaf habit between many co-occurring oaks and pines suggest different strategies for resource use, efficiency and stomatal behavior. The New Jersey Pinelands contain sandy soils with low water- and nutrient-holding capacity providing an opportunity to examine trade-offs in resource uptake and efficiency. Therefore, we compared resource use in terms of transpiration rates and leaf nitrogen content and resource-use efficiency including water-use efficiency (WUE) via gas exchange and leaf carbon isotopes and photosynthetic nitrogen-use efficiency (PNUE) between oaks (Quercus alba, Q. prinus, Q. velutina) and pines (Pinus rigida, P. echinata). We also determined environmental drivers [vapor pressure deficit (VPD), soil moisture, solar radiation] of canopy stomatal conductance (GS) estimated via sap flow and stomatal sensitivity to light and soil moisture. Net assimilation rates were similar between genera, but oak leaves used about 10% more water and pine foliage contained about 20% more N per unit leaf area. Therefore, oaks exhibited greater PNUE while pines had higher WUE based on gas exchange, although WUE from carbon isotopes was not significantly different. For the environmental drivers of GS, oaks had about 10% lower stomatal sensitivity to VPD normalized by reference stomatal conductance compared with pines. Pines exhibited a significant positive relationship between shallow soil moisture and GS, but only GS in Q. velutina was positively related to soil moisture. In contrast, stomatal sensitivity to VPD was significantly related to solar radiation in all oak species but only pines at one site. Therefore, oaks rely more heavily on groundwater resources but have lower WUE, while pines have larger leaf areas and nitrogen acquisition but lower PNUE demonstrating a trade-off between using water and nitrogen efficiently in a resource-limited ecosystem.
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Disturbance regimes within temperate forests can significantly impact carbon cycling. Additionally, projected climate change in combination with multiple, interacting disturbance effects may disrupt the capacity of forests to act as carbon sinks at large spatial and temporal scales. We used a spatially explicit forest succession and disturbance model, LANDIS-II, to model the effects of climate change, gypsy moth (Lymantria dispar L.) defoliation, and wildfire on the C dynamics of the forests of the New Jersey Pine Barrens over the next century. Climate scenarios were simulated using current climate conditions (baseline), as well as a high emissions scenario (HadCM3 A2 emissions scenario). Our results suggest that long-term changes in C cycling will be driven more by climate change than by fire or gypsy moths over the next century. We also found that simulated disturbances will affect species composition more than tree growth or C sequestration rates at the landscape level. Projected changes in tree species biomass indicate a potential increase in oaks with climate change and gypsy moth defoliation over the course of the 100-year simulation, exacerbating current successional trends towards increased oak abundance. Our research suggests that defoliation under climate change may play a critical role in increasing the variability of tree growth rates and in determining landscape species composition over the next 100 years.
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Carbono/metabolismo , Cambio Climático , Bosques , Herbivoria , Mariposas Nocturnas/fisiología , Pinus/fisiología , Quercus/fisiología , Animales , Carbono/análisis , Simulación por Computador , Monitoreo del Ambiente , Incendios , New Jersey , Nitrógeno/análisis , Nitrógeno/metabolismo , Factores de TiempoRESUMEN
In this Letter, we present the results of a hit-finding and lead optimization programme against the EP4 receptor (EP4R). In a short time period, we were able to discover five structurally diverse series of hit compounds using a combination of virtual screening methods. The most favoured hit, compound 6, was demonstrated to be a competitive antagonist of the EP4R. Compound 73 was identified following several rounds of optimization, which centred on improving both the primary EP4R affinity and selectivity against the related EP2R as well as the aqueous solubility. This work culminated in the preparation of PGN-1531, the sodium salt of 73, which showed a marked improvement in solubility (>10 mg/mL). PGN-1531 is a potent and selective antagonist at EP4Rs in vitro and in vivo, with the potential to alleviate the symptoms of migraine that result from cerebral vasodilatation.
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Descubrimiento de Drogas , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Encéfalo/irrigación sanguínea , Células HEK293 , Humanos , Ligandos , Trastornos Migrañosos/tratamiento farmacológico , Modelos Moleculares , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Vasodilatación/efectos de los fármacosRESUMEN
Oak species are well suited to water-limited conditions by either avoiding water stress through deep rooting or tolerating water stress through tight stomatal control. In co-occurring species where resources are limited, species may either partition resources in space and/or time or exhibit differing efficiencies in the use of limited resources. Therefore, this study seeks to determine whether two co-occurring oak species (Quercus prinus L. and Quercus velutina Lam.) differ in physiological parameters including photosynthesis, stomatal conductance, water-use (WUE) and nitrogen-use efficiency (NUE), as well as to characterize transpiration and average canopy stomatal responses to climatic variables in a sandy, well-drained and nutrient-limited ecosystem. The study was conducted in the New Jersey Pinelands and we measured sap flux over a 3-year period, as well as leaf gas exchange, leaf nitrogen and carbon isotope concentrations. Both oak species showed relatively steep increases in leaf-specific transpiration at low vapor pressure deficit (VPD) values before maximum transpiration rates were achieved, which were sustained over a broad range in VPD. This suggests tight stomatal control over transpiration in both species, although Q. velutina showed significantly higher leaf-level and canopy-level stomatal conductance than Q. prinus. Average daytime stomatal conductance was positively correlated with soil moisture and both oak species maintained at least 75% of their maximum canopy stomatal conductance at soil moistures in the upper soil layer (0-0.3 m) as low as 0.03 m(3) m(3)(-3). Quercus velutina had significantly higher photosynthetic rates, maximum Rubisco-limited and electron-transport-limited carboxylation rates, dark respiration rates and nitrogen concentration per unit leaf area than Q. prinus. However, both species exhibited similar WUEs and NUEs. Therefore, Q. prinus has a more conservative resource-use strategy, while Q. velutina may need to exploit niches that are locally higher in nutrients and water. Likewise, both species appear to tap deep, stable water sources, highlighting the importance of rooting depth in modeling transpiration and stomatal conductance in many oak ecosystems.
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Ecosistema , Nitrógeno/farmacología , Fósforo/farmacología , Quercus/fisiología , Agua/farmacología , Isótopos de Carbono , Gases/metabolismo , Humedad , Luz , Isótopos de Nitrógeno , Especificidad de Órganos , Fotosíntesis/efectos de los fármacos , Fotosíntesis/efectos de la radiación , Estomas de Plantas/efectos de los fármacos , Estomas de Plantas/fisiología , Estomas de Plantas/efectos de la radiación , Transpiración de Plantas/efectos de los fármacos , Transpiración de Plantas/efectos de la radiación , Quercus/efectos de los fármacos , Quercus/efectos de la radiación , Suelo , TemperaturaRESUMEN
The maximum temperature experienced by biomass during combustion has a strong effect on chemical properties of the resulting charcoal, such as sorption capacity (water and nonpolar materials) and microbial degradability. However, information about the formation temperature of natural charcoal can be difficult to obtain in ecosystems that are not instrumented prior to fires. Benzene polycarboxylic acids (BPCA) are molecular markers specific for pyrogenic carbon (PyC) which can provide information on the degree of aromatic condensation in charcoals. Here we apply the BPCA molecular marker method to a set of 10 charcoals produced during an experimental fire in a Pitch pine-scrub oak forest from litter and bark of pitch pine and inkberry plants in the Pinelands National Reserve in New Jersey, USA. We deployed temperature-sensitive crayons throughout the burn site, which recorded the maximum air temperature and made comparisons to the degree of thermal alteration recorded by BPCA molecular markers. Our results show an increase of the degree of aromatic condensation with monitored temperatures for bark biomass, while for needles no clear trend could be observed. For leaf-derived charcoals at increasing monitored fire temperatures, decreasing degree of aromatic condensation was obtained. This suggests that molecular markers can be used to roughly estimate the maximum fire temperatures experienced by bark and wood materials, but not based on leaf- and needle-derived materials. Possible applications include verifying declared pyrolysis temperatures of biochars and evaluating ecosystem fire temperature postburn.
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Biomasa , Carbón Orgánico/química , Temperatura , Termómetros , Árboles/química , Aire , Carbono/análisis , Ácidos Carboxílicos/química , Incendios , Hidrógeno/análisis , Nitrógeno/análisisRESUMEN
Lung pathology in cystic fibrosis is linked to dehydration of the airways epithelial surface which in part results from inappropriately raised sodium reabsorption through the epithelial sodium channel (ENaC). To identify a small-interfering RNA (siRNA) which selectively inhibits ENaC expression, chemically modified 21-mer siRNAs targeting human ENaCα were designed and screened. GSK2225745, was identified as a potent inhibitor of ENaCα mRNA (EC(50) (half maximal effective concentration) = 0.4 nmol/l, maximum knockdown = 85%) and protein levels in A549 cells. Engagement of the RNA interference (RNAi) pathway was confirmed using 5' RACE. Further profiling was carried out in therapeutically relevant human primary cells. In bronchial epithelial cells, GSK2225745 elicited potent suppression of ENaCα mRNA (EC(50) = 1.6 nmol/l, maximum knockdown = 82%). In human nasal epithelial cells, GSK2225745 also produced potent and long-lasting (≥72 hours) suppression of ENaCα mRNA levels which was associated with significant inhibition of ENaC function (69% inhibition of amiloride-sensitive current in cells treated with GSK2225745 at 10 nmol/l). GSK2225745 showed no evidence for potential to stimulate toll-like receptor (TLR)3, 7 or 8. In vivo, topical delivery of GSK2225745 in a lipid nanoparticle formulation to the airways of mice resulted in significant inhibition of the expression of ENaCα in the lungs. In conclusion, GSK2225745 is a potent inhibitor of ENaCα expression and warrants further evaluation as a potential novel inhaled therapeutic for cystic fibrosis.Molecular Therapy - Nucleic Acids (2013) 2, e65; doi:10.1038/mtna.2012.57; published online 15 January 2013.
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Azelastine is a selective antagonist at the human histamine-1 receptor and is used clinically in the treatment of allergic rhinitis. In this study we have investigated its duration of action in vitro in an effort to characterise the receptor and tissue components involved. Chinese hamster ovary cell membrane fragments were used to determine the kinetics of azelastine at the H1 receptor in a radioligand binding assay. Further duration of action studies were completed in tissue preparations using guinea-pig trachea and human bronchus. In radioligand binding studies, azelastine reached steady state at the H1 receptor after approximately 41 min and exhibited a significantly slower dissociation rate constant from the receptor than the first generation antihistamine, diphenhydramine. In washout studies completed in guinea-pig and human airway in vitro tissue preparations, azelastine continued to antagonise the effects of histamine at the H1 receptor for at least 18 h post-washout of the antagonist. This outcome was reversed following removal of the epithelium from guinea-pig isolated tracheal strips. These studies indicate there is a tissue component contributing to azelastine's duration of action, in addition to its direct H1 receptor binding, with evidence suggesting a role for the epithelial layer.
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Antagonistas de los Receptores Histamínicos H1/metabolismo , Antagonistas de los Receptores Histamínicos H1/farmacología , Ftalazinas/metabolismo , Ftalazinas/farmacología , Receptores Histamínicos H1/metabolismo , Animales , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Células CHO , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Humanos , Técnicas In Vitro , CinéticaRESUMEN
Although native herbivores can alter fire regimes by consuming herbaceous vegetation that serves as fine fuel and, less commonly, accumulating fuel as nest material and other structures, simultaneous considerations of contrasting effects of herbivores on fire have scarcely been addressed. We proposed that a colonial rodent, vizcacha (Lagostomus maximus), reduces and increases fire intensity at different stages in its population cycle in the semiarid scrub of Argentina. Specifically, we hypothesized that, when colonies are active, vizcachas create natural fire-breaks through intense grazing, generating over time patches of large unburned shrubs in grazed zones. In contrast, when colonies are abandoned, recovery of fine fuels and previous accumulation of coarse wood on colonies during territorial displays increases fire intensity, creating patches of high shrub mortality. To test these hypotheses, we estimated stem age of the dominant shrub (Larrea divaricata) and measured aboveground biomass in zones actively grazed by vizcachas and in ungrazed zones, and compared densities of live and dead shrubs on abandoned colonies and adjacent zones following fire. In active colonies, age and biomass of shrubs were much greater in grazed than ungrazed zones. In abandoned colonies that had been burnt, density of dead, burned shrubs was higher and density of live shrubs was lower than in adjacent zones. These results support our hypotheses and reveal a new interaction between native herbivores and fire, in which herbivores augment fire intensity by gathering fuel. Our findings indicate that, through opposing effects on fire, native herbivores enhance the heterogeneity of vegetation in woody-dominated ecosystems.
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Conducta Animal , Ecosistema , Incendios , Plantas , Roedores/psicología , Animales , Argentina , Bovinos , Dinámica PoblacionalRESUMEN
A series of potent phthalazinone-based human H(1) and H(3) bivalent histamine receptor antagonists, suitable for intranasal administration for the potential treatment of allergic rhinitis, were identified. Blockade of H(3) receptors is thought to improve efficacy on nasal congestion, a symptom of allergic rhinitis that is currently not treated by current antihistamines. Two analogues (56a and 56b) had slightly lower H(1) potency (pA(2) 9.1 and 8.9, respectively, vs 9.7 for the clinical gold-standard azelastine, and H(3) potency (pK(i) 9.6 and 9.5, respectively, vs 6.8 for azelastine). Compound 56a had longer duration of action than azelastine, low brain penetration, and low oral bioavailability, which coupled with the predicted low clinical dose, should limit the potential of engaging CNS-related side-effects associated with H(1) or H(3) antagonism.
Asunto(s)
Descubrimiento de Drogas/métodos , Ftalazinas/administración & dosificación , Ftalazinas/farmacología , Receptores Histamínicos H1/metabolismo , Receptores Histamínicos H3/metabolismo , Rinitis/tratamiento farmacológico , Administración Intranasal , Administración Oral , Antagonistas de los Receptores Histamínicos H1/administración & dosificación , Antagonistas de los Receptores Histamínicos H1/química , Antagonistas de los Receptores Histamínicos H1/farmacología , Antagonistas de los Receptores Histamínicos H1/uso terapéutico , Antagonistas de los Receptores Histamínicos H3/administración & dosificación , Antagonistas de los Receptores Histamínicos H3/química , Antagonistas de los Receptores Histamínicos H3/farmacología , Antagonistas de los Receptores Histamínicos H3/uso terapéutico , Humanos , Modelos Moleculares , Ftalazinas/química , Ftalazinas/uso terapéutico , Conformación Proteica , Receptores Histamínicos H1/químicaRESUMEN
BACKGROUND: Low density arrays (LDAs) have recently been introduced as a novel approach to gene expression profiling. Based on real time quantitative RT-PCR (QRT-PCR), these arrays enable a more focused and sensitive approach to the study of gene expression than gene chips, while offering higher throughput than more established approaches to QRT-PCR. We have now evaluated LDAs as a means of determining the expression of multiple genes simultaneously in human tissues and cells. RESULTS: Comparisons between LDAs reveal low variability, with correlation coefficients close to 1. By performing 2-fold and 10-fold serial dilutions of cDNA samples in the LDAs we determined a clear linear relationship between the gene expression data points over 5 orders of magnitude. We also showed that it is possible to use LDAs to accurately and quantitatively detect 2-fold changes in target copy number as well as measuring genes that are expressed with low and high copy numbers in the range of 1 x 10(2)-1 x 10(6) copies. Furthermore, the data generated by the LDA from a cell based pharmacological study were comparable to data generated by conventional QRT-PCR. CONCLUSION: LDAs represent a valuable new approach for sensitive and quantitative gene expression profiling.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Cartilla de ADN , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Humanos , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Trombina/farmacologíaRESUMEN
Adenosine is an endogenous nucleoside that regulates many physiological processes through the activation of its four receptors: A(1), A(2A), A(2B) and A(3). Previous studies have identified the involvement of A(2) receptors in the inhibitory activity of adenosine analogues on tumor necrosis factor-alpha (TNF-alpha) production by lipopolysaccharide (LPS) activated monocytes, but the relative contributions of A(2A) versus A(2B) receptors have not been determined in human primary monocytes. Nor has the role of A(1) and A(3) been clearly identified in the system. The lack of such information impacts on the selection of adenosine receptor agonists for disease intervention. Using LPS-stimulated human primary monocytes, we found that the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) or the A(2A) receptor agonist, 4-[2-[[6-amino-9-(N-ethyl-b-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680) produced a concentration-dependent inhibition of TNF-alpha production, with IC(50)s of 58.4nM (32.7-104.5nM, 95% confidence interval) and 49.2nM (22.7-105.9nM, 95% confidence interval), respectively. The selective A(2A) receptor blocker, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylaminso]ethyl)phenol (ZM241385, 30nM), antagonized the effects of NECA and CGS21680 (pK(B) estimates were 8.7+/-0.1 and 8.9+/-0.1, respectively), while the selective A(2B) antagonist, N-(4-cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide (MRS1754, 100nM), failed to antagonize the effects of either agonist. Furthermore, neither the A(1) receptor agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA) nor the A(3) receptor agonist, 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-d-ribofuranuronamide (2-Cl-IB-MECA) showed significant inhibitory activity at concentrations that effectively bind to their respective receptors. We conclude that A(2A) receptor activation is predominantly responsible for the inhibitory effects of adenosine receptor agonists on TNF-alpha production from LPS-stimulated monocytes.