RESUMEN
Intestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cell function. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.
Asunto(s)
Enfermedades Inflamatorias del Intestino/fisiopatología , Mesodermo/fisiología , Animales , Proliferación Celular , Colitis/genética , Colitis/fisiopatología , Colon/fisiología , Células Epiteliales/metabolismo , Fibroblastos/fisiología , Heterogeneidad Genética , Homeostasis , Humanos , Inflamación , Mucosa Intestinal/inmunología , Mucosa Intestinal/fisiología , Intestinos/inmunología , Intestinos/fisiología , Células Madre Mesenquimatosas/fisiología , Mesodermo/metabolismo , Ratones , Ratones Endogámicos C57BL , Miofibroblastos , Pericitos , Células RAW 264.7 , Factores de Transcripción SOXD/fisiología , Análisis de la Célula Individual/métodos , Tromboplastina/fisiología , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética , Vía de Señalización Wnt/fisiologíaRESUMEN
We leveraged a clinical pharmacokinetic (PK)/pharmacodynamics (PD)/efficacy relationship established with an oral phosphatidylinositol 3-kinase (PI3K)δ inhibitor (Idelalisib) in a nasal allergen challenge study to determine whether a comparable PK/PD/efficacy relationship with PI3Kδ inhibitors was observed in preclinical respiratory models of type 2 T helper cell (TH2) and type 1 T helper cell (TH1) inflammation. Results from an in vitro rat blood basophil (CD63) activation assay were used as a PD biomarker. IC50 values for PI3Kδ inhibitors, MSD-496486311, MSD-126796721, Idelalisib, and Duvelisib, were 1.2, 4.8, 0.8, and 0.5 µM. In the ovalbumin Brown Norway TH2 pulmonary inflammation model, all PI3Kδ inhibitors produced a dose-dependent inhibition of bronchoalveolar lavage eosinophils (maximum effect between 80% and 99%). In a follow-up experiment designed to investigate PK attributes [maximum (or peak) plasma concentration (Cmax), area under the curve (AUC), time on target (ToT)] that govern PI3Kδ efficacy, MSD-496486311 [3 mg/kg every day (QD) and 100 mg/kg QD] produced 16% and 93% inhibition of eosinophils, whereas doses (20 mg/kg QD, 10 mg/kg twice per day, and 3 mg/kg three times per day) produced 54% to 66% inhibition. Our profiling suggests that impact of PI3Kδ inhibitors on eosinophils is supported by a PK target with a ToT over the course of treatment close to the PD IC50 rather than strictly driven by AUC, Cmax, or Cmin (minimum blood plasma concentration) coverage. Additional studies in an Altenaria alternata rat model, a sheep Ascaris-sensitive sheep model, and a TH1-driven rat ozone exposure model did not challenge our hypothesis, suggesting that an IC50 level of TE (target engagement) sustained for 24 hours is required to produce efficacy in these traditional models. We conclude that the PK/PD observations in our animal models appear to align with clinical results associated with a TH2 airway disease.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacocinética , Enfermedades Respiratorias/tratamiento farmacológico , Enfermedades Respiratorias/inmunología , Células TH1/efectos de los fármacos , Células Th2/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/metabolismo , Masculino , Inhibidores de las Quinasa Fosfoinosítidos-3/uso terapéutico , Ratas , Enfermedades Respiratorias/metabolismoRESUMEN
The chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells (CRTH2) is a G protein-coupled receptor that has been reported to modulate inflammatory responses in various rodent models of asthma, allergic rhinitis and atopic dermatitis. In this study, we describe the biological and pharmacological properties of {(7R)-7-[[(4-fluorophenyl)sulfonyl](methyl)amino]-6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl}acetic acid (MK-7246), a novel synthetic CRTH2 antagonist. We show that MK-7246 1) has high affinity for the human, monkey, dog, rat, and mouse CRTH2, 2) interacts with CRTH2 in a reversible manner, 3) exhibits high selectivity over all prostanoid receptors as well as 157 other receptors and enzymes, 4) acts as a full antagonist on recombinant and endogenously expressed CRTH2, 5) demonstrates good oral bioavailability and metabolic stability in various animal species, 6) yields ex vivo blockade of CRTH2 on eosinophils in monkeys and sheep, and 7) significantly blocks antigen-induced late-phase bronchoconstriction and airway hyper-responsiveness in sheep. MK-7246 represents a potent and selective tool to further investigate the in vivo function of CRTH2.
Asunto(s)
Carbolinas/química , Carbolinas/farmacología , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/biosíntesis , Receptores de Prostaglandina/antagonistas & inhibidores , Receptores de Prostaglandina/biosíntesis , Células Th2/metabolismo , Animales , Perros , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Células HEK293 , Humanos , Macaca fascicularis , Ratones , Inhibidores de Agregación Plaquetaria/farmacología , Unión Proteica/inmunología , Ratas , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/fisiología , Receptores de Prostaglandina/metabolismo , Receptores de Prostaglandina/fisiología , Ovinos , Especificidad de la Especie , Células Th2/efectos de los fármacosRESUMEN
In Huntington disease, polyglutamine expansion of the protein huntingtin (Htt) leads to selective neurodegenerative loss of medium spiny neurons throughout the striatum by an unknown apoptotic mechanism. Binding of Hip-1, a protein normally associated with Htt, is reduced by polyglutamine expansion. Free Hip-1 binds to a hitherto unknown polypeptide, Hippi (Hip-1 protein interactor), which has partial sequence homology to Hip-1 and similar tissue and subcellular distribution. The availability of free Hip-1 is modulated by polyglutamine length within Htt, with disease-associated polyglutamine expansion favouring the formation of pro-apoptotic Hippi-Hip-1 heterodimers. This heterodimer can recruit procaspase-8 into a complex of Hippi, Hip-1 and procaspase-8, and launch apoptosis through components of the 'extrinsic' cell-death pathway. We propose that Htt polyglutamine expansion liberates Hip-1 so that it can form a caspase-8 recruitment complex with Hippi. This novel non-receptor-mediated pathway for activating caspase-8 might contribute to neuronal death in Huntington disease.
Asunto(s)
Proteínas Portadoras/metabolismo , Caspasas/metabolismo , Proteínas de Unión al ADN , Enfermedad de Huntington/metabolismo , Neuronas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Apoptosis/fisiología , Proteínas Portadoras/genética , Caspasa 8 , Caspasa 9 , Caspasas/genética , Células Cultivadas , Activación Enzimática , Humanos , Proteína Huntingtina , Enfermedad de Huntington/enzimología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/ultraestructura , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estructura Terciaria de Proteína , Ratas , Alineación de Secuencia , Distribución Tisular , Técnicas del Sistema de Dos HíbridosRESUMEN
A new series of indole amide acting as hCRTH2 receptor ligands had been explored and are described herein. Several amide derivatives displaying low nanomolar activity in hCRTH2 binding and whole blood assays were identified. They were found to behave as a full antagonists, exhibiting good selectivity over related prostaglandin receptors. Also, prototypical compounds in this novel series which displayed acceptable CYP profiles and were orally bioavailable in rats were identified.
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Amidas/síntesis química , Amidas/farmacología , Indoles/síntesis química , Indoles/farmacología , Receptores de Dopamina D2/agonistas , Amidas/química , Animales , Indoles/química , Concentración 50 Inhibidora , Ligandos , Estructura Molecular , Unión Proteica/efectos de los fármacos , RatasRESUMEN
A new class of 7-azaindole analogs of MK-7246 as potent and selective CRTH2 antagonists is reported. The SAR leading to the identification of the optimal azaindole regioisomer as well as the pharmacokinetics and off-target activities of the most potent antagonists are disclosed.
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Indoles/química , Indoles/farmacología , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/antagonistas & inhibidores , Receptores de Prostaglandina/metabolismo , Animales , Carbolinas/química , Humanos , Indoles/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
In this manuscript we wish to report the discovery of MK-7246 (4), a potent and selective CRTH2 (DP2) antagonist. SAR studies leading to MK-7246 along with two synthetic sequences enabling the preparation of this novel class of CRTH2 antagonist are reported. Finally, the pharmacokinetic and metabolic profile of MK-7246 is disclosed.
Asunto(s)
Carbolinas/química , Enfermedades Pulmonares/tratamiento farmacológico , Receptores Inmunológicos/antagonistas & inhibidores , Receptores de Prostaglandina/antagonistas & inhibidores , Animales , Carbolinas/farmacocinética , Carbolinas/uso terapéutico , Humanos , Macaca mulatta , Microsomas Hepáticos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Relación Estructura-ActividadRESUMEN
Chronic obstructive pulmonary disease (COPD) is a prevalent pulmonary disease characterized by a progressive decline in lung function. The identification of biomarkers capable of predicting the rate of lung function decline or capable of giving an early read on drug efficacy in clinical trials would be very useful. The aim of this study was to identify plasma biomarkers capable of accurately distinguishing patients with COPD from healthy controls. Eighty-nine plasma markers in 40 COPD patients and 20 healthy smoker controls were analyzed. The COPD patients were divided into two subgroups, rapid and slow decliners based on their rate of lung function decline measured over 15 years. Univariate analysis revealed that 25 plasma markers were statistically different between rapid decliners and controls, 4 markers were different between slow decliners and controls, and 10 markers were different between rapid and slow decliners (p < 0.05). Multivariate analysis led to the identification of groups of plasma markers capable of distinguishing rapid decliners from controls (signature 1), slow decliners from controls (signature 2) and rapid from slow decliners (signature 3) with over 90% classification accuracy. Importantly, signature 1 was shown to be longitudinally stable using plasma samples taken a year later from a subset of patients. This study describes a novel set of plasma markers differentiating slow from rapid decline of lung function in COPD. If validated in distinct and larger cohorts, the signatures identified will have important implications in both disease diagnosis, as well as the clinical evaluation of new therapies.
Asunto(s)
Biomarcadores/sangre , Enfermedad Pulmonar Obstructiva Crónica/sangre , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Estudios de Casos y Controles , Femenino , Volumen Espiratorio Forzado/fisiología , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Índice de Severidad de la Enfermedad , Factores de TiempoRESUMEN
Paget's disease of bone (PDB) is a common chronic bone disorder. In the French-Canadian population, the p.Pro392Leu mutation within the SQSTM1 gene is involved in 46% of familial forms. In New Zealand, the emergence of PDB in offspring inheriting SQSTM1 mutations was reported to be delayed by a decade compared to their parents. We aimed at assessing the clinical phenotype of offspring carriers of this mutation in our French-Canadian cohort. We reviewed research records from adult offspring carriers of this mutation aged <90 years and their affected parents. In parents, we collected data on sex, age at diagnosis, number of affected bones, total serum alkaline phosphatase levels (tALPs) at diagnosis. In offspring, PDB extended phenotype assessment relying on tALPs, bone specific alkaline phosphatase levels (bALPs), procollagen type 1 amino-terminal propeptide (P1NP), whole body bone scan and skull and pelvis radiographs, was performed at inclusion from 1996 to 2009 and updated in 2016 to 2018, if not done during the past 8 years. The results showed that among the 36 offspring with an updated phenotype, four of them developed a clinical phenotype of PDB characterized by monostotic or polyostotic increased bone uptake associated with typical radiographic lesions in the affected sites, representing an incidence of 1.83 per 1000 person-years. Moreover, the age at PDB diagnosis was delayed by at least 10 years in the adult offspring carriers of the p.Pro392Leu mutation versus their affected parents. Our findings support the utility of a regular monitoring of the adult offspring without PDB but carriers of this mutation.
RESUMEN
Chronic obstructive pulmonary disease (COPD) is characterized by the degradation of elastin, the major insoluble protein of lung tissues. The degradation of elastin gives rise to desmosine (DES) and isodesmosine (IDES), two major urinary products typified by a hydrophilic pyridinium-based cross-linker structure. A high sensitivity method based on nanoflow liquid chromatography tandem mass spectrometry with multiple reaction monitoring was developed for the analysis of urinary DES and IDES. The analytes were derivatized with propionic anhydride and deuterated DES (D(4)-DES) was used as an internal standard. This method enables the quantification of DES and IDES in as little as 50 microL of urine and provides a detection limit of 0.10 ng/mL (0.95 fmol on-column). We report the analysis of DES and IDES in a cohort of 40 urine specimens from four groups of individuals: (a) COPD rapid decliners (11.8 +/- 3.7 ng/mg creatine (crea)), (b) COPD slow decliners (16.0 +/- 3.1 ng/mg crea), (c) healthy smokers (13.2 +/- 1.9 ng/mg crea), and (d) healthy nonsmokers (14.9 +/- 2.9 ng/mg crea). Our analysis reveals a statistically significant decrease in the level of urinary DES and IDES in COPD rapid decliner patients compared to healthy nonsmoker controls and COPD slow decliner patients. This methodology may be useful for monitoring DES and IDES levels in well controlled animal models for COPD or for longitudinal studies in COPD patients.
Asunto(s)
Cromatografía Liquida/métodos , Desmosina/orina , Isodesmosina/orina , Límite de Detección , Espectrometría de Masas/métodos , Espectrometría de Masas en Tándem/métodos , Elastina/análisis , HumanosRESUMEN
We describe herein a novel series of 3-amino-4-hydrazine-cyclobut-3-ene-1,2-diones as potent and selective inhibitors against the CXCR2 chemokine receptor and IL-8-mediated chemotaxis of a CXCR2-expressing cell line. Furthermore, these alkyl-hydrazine series inhibitors such as 5b demonstrated acceptable metabolic stability when incubated in human and rat microsomes.
Asunto(s)
Antiinflamatorios/síntesis química , Hidrazinas/síntesis química , Receptores de Interleucina-8B/antagonistas & inhibidores , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Células CHO , Quimiotaxis/efectos de los fármacos , Cricetinae , Cricetulus , Diseño de Fármacos , Humanos , Hidrazinas/química , Hidrazinas/farmacología , Interleucina-8/metabolismo , Microsomas Hepáticos/metabolismo , Ratas , Receptores de Interleucina-8B/metabolismo , Relación Estructura-ActividadRESUMEN
Recent experiments have called into question the assumption of a random A-site distribution in mixed-valence colossal magnetoresistance (CMR) manganites. We explored the possibility of an A-site (La(3+)/Sr(2+)) ordering in a CMR manganite La(3/4)Sr(1/4)MnO(3) using molecular dynamics (MD) simulations with a newly developed force field (FF) and quantum mechanics (QM) (density functional theory with the generalized gradient approximation) calculations of the relative stability of structures obtained from MD. Both methods suggest that the degree of stabilization (enthalpy gain) of A-site ordering is not sufficient to overcome the accompanying entropy loss, supporting the assumption of a random A-site distribution in La(3/4)Sr(1/4)MnO(3). This approach combining MD and QM as well as the versatile FF developed in this study should be useful for investigating the structure and functionality of magnetic tunnel junction devices involving composite materials of mixed-valence manganites.
RESUMEN
A novel series of tricyclic tetrahydroquinolines were identified as potent and selective CRTh2 receptor antagonists. The agonism and antagonism switch was achieved through structure-based drug design (SBDD) using a CRTh2 receptor homologue model. The challenge of very low exposures in pharmacokinetic studies was overcome by exhaustive medicinal chemistry lead optimization through focused SAR studies on the tricyclic core. Further optimization resulted in the identification of the preclinical candidate 4-(cyclopropyl((3aS,9R,9aR)-7-fluoro-4-(4-(trifluoromethoxy)benzoyl)-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]quinolin-9-yl)amino)-4-oxobutanoic acid (15c, MK-8318) with potent and selective CRTh2 antagonist activity and a favorable PK profile suitable for once daily oral dosing for potential treatment of asthma.
RESUMEN
The anaphylatoxin C3a is an important immune regulator with a number of distinct functions in both innate and adaptive immunity. Many of these roles have been ascribed to C3a based on studies in mice genetically modified to lack its precursor, C3, or its receptor, C3aR. However, other presumed functions of C3a are based on results obtained with a recently described small molecule ligand of C3aR, SB 290157. Although this compound was originally described as an antagonist and appears to act as such in some systems, it has recently been shown to have effects that cannot be explained by simple antagonism of C3aR. In the current study, SB 290157 is shown to have full agonist activity on C3aR in a variety of cell systems, including a calcium mobilization assay in transfected RBL cells, a beta-lactamase assay in CHO-NFAT-bla-Galpha(16) cells and an enzyme-release assay in differentiated U-937 cells. On the other hand, the compound lacks agonist activity in guinea pig platelets, cells known to express C3aR at very low levels. SB 290157 agonism of C3aR is consistent with recent discrepant data obtained using this molecule. These results caution against attributing novel roles to C3a based on data obtained with SB 290157 and highlight a continuing need for the identification of true small molecule C3aR antagonists.
Asunto(s)
Arginina/análogos & derivados , Compuestos de Bencidrilo/farmacología , Calcio/metabolismo , Proteínas de la Membrana/agonistas , Receptores de Complemento/agonistas , Animales , Arginina/farmacología , Unión Competitiva , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Células CHO , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Complemento C3a , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Humanos , Macaca fascicularis , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratas , Receptores de Complemento/antagonistas & inhibidores , Receptores de Complemento/genética , Transfección , Células U937 , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
INTRODUCTION: Whole blood functional assays are pharmacologically relevant in the drug discovery process to evaluate potency in a relevant biological matrix, to support establishment of PK/PD relationships and to aid in human dose predictions. However development of B cell activation assays by BCR ligation in rat whole blood has not been previously described. The aim of the present study was to develop novel methods of B cell activation in rat whole blood. METHODS: B cell activation in rat whole blood was evaluated by measuring CD86 up-regulation via flow cytometry. Rat B cells in whole blood were stimulated with dextran-coupled anti-IgD or a combination of anti-IgD and TLR9 agonist. BTK, SYK, and PI3Kδ inhibitors were added to rat whole blood prior to activation with dextran-coupled anti-IgD or anti-IgD and TLR9 agonist combination for pharmacological validation of the assay. RESULTS: Both methods of stimulation in rat whole blood evoked robust B cell activation in a uni-modal fashion. Highly selective inhibitors of BTK, SYK, and PI3Kδ dose-dependently attenuated B cell activity evoked by both dextran-coupled anti-IgD and combined anti-IgD and TLR9 agonist. Compound potencies and rank order determined by the two assays were comparable. DISCUSSION: Two novel methods were developed to stimulate B cells in rat whole blood, that have the potential to be used to support drug discovery efforts in the therapeutic targeting of B cells. Furthermore, we pharmacologically validated these whole blood assays using highly selective inhibitors of BTK, SYK, and PI3Kδ, signaling kinases which are downstream of the B cell receptor.
Asunto(s)
Linfocitos B/efectos de los fármacos , Sangre/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Sangre/inmunología , Relación Dosis-Respuesta a Droga , Femenino , Inhibidores de Proteínas Quinasas/química , Ratas , Ratas Endogámicas Lew , Relación Estructura-ActividadRESUMEN
1. The recombinant human prostaglandin D(2) (PGD(2)) receptor, hCRTH2, has been expressed in HEK293(EBNA) and characterized with respect to radioligand binding and signal transduction properties. High and low affinity binding sites for PGD(2) were identified in the CRTH2 receptor population by saturation analysis with respective equilibrium dissociation constants (K(D)) of 2.5 and 109 nM. This revealed that the affinity of PGD(2) for CRTH2 is eight times less than its affinity for the DP receptor. 2. Equilibrium competition binding assays revealed that of the compounds tested, only PGD(2) and several related metabolites bound with high affinity to CRTH2 (K(i) values ranging from 2.4 to 34.0 nM) with the following rank order of potency: PGD(2)>13,14-dihydro-15-keto PGD(2)>15-deoxy-Delta(12,14)-PGJ(2)>PGJ(2)>Delta(12)-PGJ(2)>15(S)-15 methyl-PGD(2). This is in sharp contrast with the rank order of potency obtained at DP : PGD(2)>PGJ(2)>Delta(12)-PGJ(2)>15-deoxy-Delta(12,14)-PGJ(2) >>>13,14-dihydro-15-keto-PGD(2). 3. Functional studies demonstrated that PGD(2) activation of recombinant CRTH2 results in decrease of intracellular cAMP in a pertussis toxin-sensitive manner. Therefore, we showed that CRTH2 can functionally couple to the G-protein G(alphai/o). PGD(2) and related metabolites were tested and their rank order of potency followed the results of the membrane binding assay. 4. By Northern blot analysis, we showed that, besides haemopoietic cells, CRTH2 is expressed in many other tissues such as brain, heart, thymus, spleen and various tissues of the digestive system. In addition, in situ hybridization studies revealed that CRTH2 mRNA is expressed in human eosinophils. Finally, radioligand binding studies demonstrated that two eosinophilic cell lines, butyric acid-differentiated HL-60 and AML 14.3D10, also endogenously express CRTH2.
Asunto(s)
Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Unión Competitiva/efectos de los fármacos , Unión Competitiva/fisiología , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Células HL-60 , Humanos , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismo , Receptores Inmunológicos/agonistas , Receptores Inmunológicos/biosíntesis , Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/biosíntesis , Receptores de Prostaglandina/fisiología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , TransfecciónRESUMEN
BACKGROUND: Prostaglandin D2 (PGD2) is released from mast cells during the allergic response. OBJECTIVE: Since PGD2 has been shown to induce nasal congestion in humans, we investigated the distribution of hematopoietic prostaglandin D synthase (PGDS) and the two PGD2 receptors, DP and CRTH2 in human nasal mucosa from healthy subjects and subjects suffering from polyposis, a severe form of chronic rhinosinusitis. METHODS: DP mRNA expression was detected by in situ hybridization while PGDS, CRTH2 and various leukocyte markers expression were revealed by immunohistochemistry. RESULTS: In the normal mucosa, PGDS was only detected in few resident mast cells while CRTH2 was undetectable. In contrast, DP receptor mRNA was detected in epithelial goblet cells, serous glands and in the vasculature. In the nasal mucosa of subjects suffering from polyposis: (1) PGDS was detected in mast cells and other large infiltrating inflammatory cells, (2) both DP mRNA and CRTH2 were detected in eosinophils and (3) CRTH2 was detected on a subset of infiltrating T cells. Although DP mRNA could not be detected in the T cells invading the nasal mucosa, it was found to be expressed in the T cells present in the lymph node and the thymus from normal individuals. CONCLUSION: This study indicates that cells capable of producing PGD2 are present in the nasal mucosa and that both PGD2 receptors, DP and CRTH2, might play a role in inflammatory disease of the upper airways.
Asunto(s)
Oxidorreductasas Intramoleculares/biosíntesis , Mastocitos/metabolismo , Mucosa Nasal/metabolismo , Pólipos Nasales/metabolismo , Receptores Inmunológicos/biosíntesis , Receptores de Prostaglandina/biosíntesis , Adulto , Anciano , Eosinófilos/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Hipersensibilidad/metabolismo , Hipersensibilidad/patología , Hibridación in Situ , Inflamación/metabolismo , Inflamación/patología , Lipocalinas , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Masculino , Persona de Mediana Edad , Mucosa Nasal/citología , Mucosa Nasal/patología , Pólipos Nasales/patología , ARN Mensajero/biosíntesis , Linfocitos T/metabolismo , Timo/citología , Timo/metabolismoRESUMEN
Alternaria alternata is a fungal allergen linked to the development of severe asthma in humans. In view of the clinical relationship between A. alternata and asthma, we sought to investigate the allergic activity of this antigen after direct application to the lungs of Brown Norway rats. Here we demonstrate that a single intratracheal instillation of A. alternata induces dose and time dependent eosinophil influx, edema and Type 2 helper cell cytokine production in the lungs of BN rats. We established the temporal profile of eosinophilic infiltration and cytokine production, such as Interleukin-5 and Interleukin-13, following A. alternata challenge. These responses were comparable to Ovalbumin induced models of asthma and resulted in peak inflammatory responses 48h following a single challenge, eliminating the need for multiple sensitizations and challenges. The initial perivascular and peribronchiolar inflammation preceded alveolar inflammation, progressing to a more sub-acute inflammatory response with notable epithelial cell hypertrophy. To limit the effects of an A. alternata inflammatory response, MK-7246 was utilized as it is an antagonist for Chemoattractant Receptor-homologous molecule expressed in Th2 cells. In a dose-dependent manner, MK-7246 decreased eosinophil influx and Th2 cytokine production following the A. alternata challenge. Furthermore, therapeutic administration of corticosteroids resulted in a dose-dependent decrease in eosinophil influx and Th2 cytokine production. Reproducible asthma-related outcomes and amenability to pharmacological intervention by mechanisms relevant to asthma demonstrate that an A. alternata induced pulmonary inflammation in BN rats is a valuable preclinical pharmacodynamic in vivo model for evaluating the pharmacological inhibitors of allergic pulmonary inflammation.
Asunto(s)
Alternaria/efectos de los fármacos , Antiinflamatorios/farmacología , Carbolinas/farmacología , Neumonía/tratamiento farmacológico , Receptores de Formil Péptido/metabolismo , Células Th2/efectos de los fármacos , Alérgenos/inmunología , Alternaria/inmunología , Animales , Asma/tratamiento farmacológico , Asma/inmunología , Asma/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Eosinófilos/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Interleucina-13/inmunología , Interleucina-13/metabolismo , Interleucina-5/inmunología , Interleucina-5/metabolismo , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/metabolismo , Masculino , Ovalbúmina/inmunología , Ovalbúmina/farmacología , Neumonía/inmunología , Neumonía/metabolismo , Ratas , Ratas Endogámicas BN , Receptores de Formil Péptido/inmunología , Células Th2/inmunologíaRESUMEN
The formin homology (FH) proteins play a crucial role in cytoskeleton remodelling during many essential processes. In this study, we demonstrate for the first time that the formin-homology-domain-containing protein FHOD1 is cleaved by caspase-3 at the SVPD(616) site during apoptosis. Using confocal microscopy, we further demonstrate that while full length FHOD1 is mostly cytoplasmic, the FHOD1 N-terminal cleavage product is diffusely localized throughout the cytoplasm and the nucleoplasm, whereas the C-terminal cleavage product is almost exclusively nuclear with some nucleolar localization. Finally, using a run-on transcription assay we show that the C-terminal FHOD1 cleavage product has the ability to inhibit RNA polymerase I transcription when overexpressed in HeLa cells as shown by blockage of BrUTP incorporation.
Asunto(s)
Apoptosis , Caspasa 3/metabolismo , Nucléolo Celular/metabolismo , Proteínas Fetales/metabolismo , Proteínas Nucleares/metabolismo , ARN Ribosómico/genética , Secuencias de Aminoácidos , Caspasa 3/genética , Línea Celular Transformada , ADN Complementario , Proteínas Fetales/química , Forminas , Regulación de la Expresión Génica , Células HeLa , Humanos , Proteínas Nucleares/química , ARN Polimerasa I/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes , Transcripción Genética , TransfecciónRESUMEN
The chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells (CRTH2) is a G protein-coupled receptor whose function in vivo has been incompletely characterized. One of the reasons is that its current known ligands, prostaglandin D(2) and some of its metabolites, have either poor selectivity for CRTH2 or are metabolically unstable in vivo. In this study, we describe the biological and pharmacological properties of L-888,607, the first synthetic potent and selective CRTH2 agonist. We show that L-888,607 exhibits 1) subnanomolar affinity for the human CRTH2 receptor, 2) high selectivity over all other prostanoid receptors and other receptors tested, 3) agonistic activity on recombinant and endogenously expressed CRTH2 receptor, and 4) relative stability in vivo. L-888,607 thus represents a suitable tool to investigate the in vivo function of CRTH2.