RESUMEN
The canine cytokine IL-31 induces pruritus in dogs and can be detected in dogs with atopic dermatitis; however very little is understood around its interactions with specific canine cells. We hypothesize that IL-31 is involved in the progression of allergic skin disease by coordinating the interaction between the immune system with skin and neuronal systems. The goal of the following work was to identify cells that produce IL-31 as well as cells that may respond to this cytokine. Peripheral blood mononuclear cells (PBMCs) were collected from naïve and house dust mite (HDM) allergen-sensitized beagle dogs and used for ex vivo characterization of cytokine production assessed using ELISpot and quantitative immunoassay. Sensitization to HDM allergen induced a T-helper type 2 (Th2) cell phenotype characterized by an increase in the production of IL-4 protein. Interestingly, repeated allergen challenge over time also resulted in an increase in IFN-γ. Further evaluation showed that co-stimulation of Th2 polarized cells with antigen and the bacterial component Staphylococcus enterotoxin B (SEB) produced higher levels of IL-31 compared to either stimulant alone. Production of IL-31 when PBMCs were stimulated by T cell mitogens suggests T cells as a source of IL-31. Quantitative real-time PCR was utilized to determine expression of the IL-31 receptor alpha chain in canine cell lines and tissue. Canine monocytic cells, keratinocytes, and dorsal root ganglia were shown to express the IL-31 receptor alpha chain mRNA. In a multifaceted disease such as canine atopic dermatitis, the combination of Th2 polarization and microbial presence may lead to IL-31 mediated effects driving inflammation and pruritus by immune cells, keratinocytes, and direct neuronal stimulation.
Asunto(s)
Antígenos Dermatofagoides/inmunología , Dermatitis Atópica/veterinaria , Enfermedades de los Perros/inmunología , Interleucinas/inmunología , Células Th2/inmunología , Animales , Dermatitis Atópica/inmunología , Perros , Ensayo de Inmunoadsorción Enzimática/veterinaria , Interferón gamma/sangre , Interferón gamma/inmunología , Interleucina-4/sangre , Interleucina-4/inmunología , Leucocitos Mononucleares , ARN/química , ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinariaRESUMEN
BACKGROUND: Interleukin-31 (IL-31) is a member of the gp130/interleukin-6 cytokine family that is produced by cell types such as T helper 2 lymphocytes and cutaneous lymphocyte antigen positive skin homing T cells. When overexpressed in transgenic mice, IL-31 induces severe pruritus, alopecia and skin lesions. In humans, IL-31 serum levels correlate with the severity of atopic dermatitis in adults and children. HYPOTHESIS/OBJECTIVE: To determine the role of IL-31 in canine pruritus and naturally occurring canine atopic dermatitis (AD). ANIMALS: Purpose-bred beagle dogs were used for laboratory studies. Serum samples were obtained from laboratory animals, nondiseased client-owned dogs and client-owned dogs diagnosed with naturally occurring AD. METHODS: Purpose-bred beagle dogs were administered canine interleukin-31 (cIL-31) via several routes (intravenous, subcutaneous or intradermal), and pruritic behaviour was observed/quantified via video monitoring. Quantitative immunoassay techniques were employed to measure serum levels of cIL-31 in dogs. RESULTS: Injection of cIL-31 into laboratory beagle dogs caused transient episodes of pruritic behaviour regardless of the route of administration. When evaluated over a 2 h period, dogs receiving cIL-31 exhibited a significant increase in pruritic behaviour compared with dogs that received placebo. In addition, cIL-31 levels were detectable in 57% of dogs with naturally occurring AD (≥ 13 pg/mL) but were below limits of quantification (<13 pg/mL) in normal, nondiseased laboratory or client-owned animals. CONCLUSIONS: Canine IL-31 induced pruritic behaviours in dogs. Canine IL-31 was detected in the majority of dogs with naturally occurring AD, suggesting that this cytokine may play an important role in pruritic allergic skin conditions, such as atopic dermatitis, in this species.
Asunto(s)
Dermatitis Atópica/veterinaria , Enfermedades de los Perros/metabolismo , Interleucinas/farmacología , Prurito/veterinaria , Animales , Línea Celular , Clonación Molecular , Dermatitis Atópica/metabolismo , Perros , Regulación de la Expresión Génica/fisiología , Interleucinas/metabolismo , Monocitos/metabolismo , Prurito/inducido químicamente , Transducción de SeñalRESUMEN
A series of long (11-15) hydrocarbon chain diols and diacids with various central functional groups and terminal gem-dimethyl or -methyl/aryl substituents was synthesized and evaluated in both in vivo and in vitro assays for its potential to favorably alter lipid disorders including metabolic syndrome. Compounds were assessed for their effects on the de novo incorporation of radiolabeled acetate into lipids in primary cultures of rat hepatocytes, as well as for their effects on lipid and glycemic variables in obese female Zucker fatty rats, Crl:(ZUC)-faBR. The most active compounds were hydroxyl-substituted symmetrical diacids and diols with a 13-atom chain and terminal gem-dimethyl substituents. Furthermore, biological activity was enhanced by central substitution with O, C=O, S, S=O compared to the methylene analogues and was diminished for compounds with central functional groups such as carbamate, ester, urea, acetylmethylene, and hydroxymethylene.
Asunto(s)
Alcoholes/uso terapéutico , Diabetes Mellitus Experimental/tratamiento farmacológico , Ácidos Dicarboxílicos/uso terapéutico , Hidrocarburos/uso terapéutico , Hiperlipidemias/tratamiento farmacológico , Hipolipemiantes/uso terapéutico , Administración Oral , Alcoholes/administración & dosificación , Alcoholes/síntesis química , Animales , Diabetes Mellitus Experimental/metabolismo , Ácidos Dicarboxílicos/administración & dosificación , Ácidos Dicarboxílicos/síntesis química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Tolerancia a Medicamentos , Femenino , Hepatocitos/efectos de los fármacos , Hidrocarburos/administración & dosificación , Hidrocarburos/síntesis química , Hiperlipidemias/metabolismo , Hipolipemiantes/administración & dosificación , Hipolipemiantes/síntesis química , Técnicas In Vitro , Lípidos/antagonistas & inhibidores , Lípidos/biosíntesis , Estructura Molecular , Ratas , Ratas Zucker , Relación Estructura-Actividad , Factores de TiempoRESUMEN
Recent studies indicating that some nonsteroidal anti-inflammatory drugs (NSAIDs) selectively modulate gamma-secretase cleavage of amyloid precursor protein (APP) while sparing Notch processing have generated interest in discovery of novel gamma-secretase modulators with the "NSAID-like" efficacy profile. The objective of the present studies was to compare the efficacy of a subset of NSAIDs with previously reported classical gamma-secretase inhibitors LY-411575 [N(2)-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N(1)-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide]and DAPT [N-[N- (3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester] in Tg2576 mice. Flurbiprofen (10 and 25 mg/kg/day) was overtly toxic and elicited significant (but nonselective) reductions in both Abeta(1-40) and Abeta(1-42) in the plasma in one of two studies. Flurbiprofen also produced a small reduction in Abeta(1-40) in the cortex at 25 mg/kg/day but did not affect Abeta levels in hippocampus or cerebrospinal fluid. Ibuprofen and sulindac sulfide were neither overtly toxic nor efficacious at doses up to 50 mg/kg/day. The effects of NSAIDs LY-411575 and DAPT were tested in guinea pig embryonic neuronal cultures to determine whether the selective reductions in Abeta(1-42) observed in cell lines overexpressing human mutant APP can be reproduced in a neuronal model of physiological Abeta production and secretion. Flurbiprofen and sulindac nonselectively reduced Abeta(1-40) and Abeta(1-42) at concentrations > or =125 microM, although cytotoxicity was noted at > or =250 microM sulindac. Ibuprofen had no effect at concentrations up to 500 microM. In contrast, DAPT and LY-411575 potently and completely inhibited Abeta(1-40), Abeta(1-42), and Abeta(1-38) in the absence of cytotoxicity. The divergence of the present data from published reports raises the need to examine the conditions necessary to perceive selective Abeta(1-42) reduction by NSAIDs in neuronal tissue.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Dipéptidos/farmacología , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/líquido cefalorraquídeo , Animales , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Cobayas , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/líquido cefalorraquídeo , Placa AmiloideRESUMEN
We have identified a novel omega-hydroxy-alkanedicarboxylic acid, ESP 55016, that favorably alters serum lipid variables in obese female Zucker (fa/fa) rats. ESP 55016 reduced serum non-HDL-cholesterol (non-HDL-C), triglyceride, and nonesterified fatty acid levels while increasing serum HDL-C and beta-hydroxybutyrate levels in a dose-dependent manner. ESP 55016 reduced fasting serum insulin and glucose levels while also suppressing weight gain. In primary rat hepatocytes, ESP 55016 increased the oxidation of [(14)C]palmitate in a dose- and carnitine palmitoyl transferase-I (CPT-I)-dependent manner. Furthermore, in primary rat hepatocytes and in vivo, ESP 55016 inhibited fatty acid and sterol synthesis. The "dual inhibitor" activity of ESP 55016 was unlikely attributable to the activation of the AMP-activated protein kinase (AMPK) pathway because AMPK and acetyl-CoA carboxylase (ACC) phosphorylation states as well as ACC activity were not altered by ESP 55016. Further studies indicated the conversion of ESP 55016 to a CoA derivative in vivo. ESP 55016-CoA markedly inhibited the activity of partially purified ACC. The activity of partially purified HMG-CoA reductase was not altered by the xenobiotic-CoA. These data suggest that ESP 55016-CoA favorably alters lipid metabolism in a model of diabetic dyslipidemia in part by initially inhibiting fatty acid and sterol synthesis plus enhancing the oxidation of fatty acids through the ACC/malonyl-CoA/CPT-I regulatory axis.
Asunto(s)
Hiperlipidemias/tratamiento farmacológico , Lípidos/antagonistas & inhibidores , Síndrome Metabólico/tratamiento farmacológico , Proteínas Quinasas Activadas por AMP , Animales , Glucemia/efectos de los fármacos , Células Cultivadas , Coenzima A , Ácidos Dicarboxílicos/farmacología , Ácidos Dicarboxílicos/uso terapéutico , Relación Dosis-Respuesta a Droga , Ácidos Grasos/biosíntesis , Femenino , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Insulina/sangre , Peroxidación de Lípido/efectos de los fármacos , Lípidos/biosíntesis , Complejos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Zucker , Esteroles/biosíntesis , Aumento de Peso/efectos de los fármacosRESUMEN
Nicastrin is a component of the gamma-secretase complex that has been shown to adhere to presenilin-1 (PS1), Notch, and APP. Here we demonstrate that Nicastrin-deficient mice showed a phenotype that is indistinguishable from PS1/PS2 double knock-out mice, whereas heterozygotes were healthy and viable. Fibroblasts derived from Nicastrin-deficient embryos were unable to generate amyloid beta-peptide and failed to release the intracellular domain of APP- or Notch1-Gal4-VP16 fusion proteins. Additionally, C- and N-terminal fragments of PS1 and the C-terminal fragments of PS2 were not detectable in Nicastrin-null fibroblasts, whereas full-length PS1 accumulated in null fibroblasts, indicating that Nicastrin is required for the endoproteolytic processing of presenilins. Interestingly, cells derived from Nicastrin heterozygotes produced relatively higher levels of amyloid beta-peptide whether the source was endogenous mouse or transfected human APP. These data demonstrate that Nicastrin is essential for the gamma-secretase cleavage of APP and Notch in mammalian cells and that Nicastrin has both positive and negative functions in the regulation of gamma-secretase activity.
Asunto(s)
Endopeptidasas/metabolismo , Regulación Enzimológica de la Expresión Génica , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Adenoviridae/genética , Alelos , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas , Membrana Celular/metabolismo , Medios de Cultivo Condicionados/farmacología , ADN Complementario/metabolismo , Fibroblastos/metabolismo , Genes Reporteros , Genotipo , Proteínas Fluorescentes Verdes , Heterocigoto , Humanos , Luciferasas/metabolismo , Proteínas Luminiscentes/metabolismo , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/fisiología , Ratones , Ratones Noqueados , Péptidos/química , Fenotipo , Presenilina-1 , Presenilina-2 , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Factores de TiempoRESUMEN
The time dependency of the spontaneous aggregation of the fibrillogenic beta-amyloid peptide, Abeta1-40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-Abeta antibody 6E10, raised against residues 1-17, at concentrations of 200-300 nM delayed significantly the aggregation of 50 microM amyloid peptide. The anti-Abeta antibody 4G8, raised against residues 17-24, was much less active in that respect, while the antibody A162, raised against the C-terminal residues 39-43 of the full-length Abeta was totally inactive at those concentrations. Concomitant with the aggregation experiments, we also measured the time dependency of the Abeta1-40-induced toxicity toward SH-EPI cells and hippocampal neurons, evaluated by SYTOX Green fluorescence, lactate dehydrogenase release, and activation of caspases. The extent of cell damage measured by all methods reached a maximum at the same time and this maximum coincided with that of the concentration of AM. According to the kinetic scheme, the latter is the only transient peptide species whose concentration passes through a maximum. Thus, it appears that the toxic species of Abeta1-40 is most likely the same transient activated monomer that is responsible for the nucleation of fibril formation. These conclusions should provide a structural basis for understanding the toxicity of Abeta1-40 in vitro and possibly in vivo.