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1.
Bioorg Med Chem ; 27(4): 579-588, 2019 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-30626555

RESUMEN

The antioxidant natural product sulforaphane (SFN) is an oil with poor aqueous and thermal stability. Recent work with SFN has sought to optimize methods of formulation for oral and topical administration. Herein we report the design of new analogs of SFN with the goal of improving stability and drug-like properties. Lead compounds were selected based on potency in a cellular screen and physicochemical properties. Among these, 12 had good aqueous solubility, permeability and long-term solid-state stability at 23 °C. Compound 12 also displayed comparable or better efficacy in cellular assays relative to SFN and had in vivo activity in a mouse cigarette smoke challenge model of acute oxidative stress.


Asunto(s)
Antioxidantes/farmacología , Ciclobutanos/farmacología , Descubrimiento de Drogas , Isotiocianatos/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Antioxidantes/síntesis química , Antioxidantes/farmacocinética , Línea Celular , Ciclobutanos/síntesis química , Ciclobutanos/farmacocinética , Expresión Génica , Hemo-Oxigenasa 1/genética , Humanos , Isotiocianatos/síntesis química , Isotiocianatos/farmacocinética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Ratones Endogámicos C57BL , Estructura Molecular , Estrés Oxidativo/efectos de los fármacos , Ratas , Solubilidad , Relación Estructura-Actividad , Sulfóxidos , Tiocarbamatos/síntesis química , Tiocarbamatos/farmacocinética , Tiocarbamatos/farmacología
2.
Am J Physiol Lung Cell Mol Physiol ; 313(2): L305-L312, 2017 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-28473321

RESUMEN

During lung inflation, airspace dimensions are affected nonlinearly by both alveolar expansion and recruitment, potentially confounding the identification of emphysematous lung by hyperpolarized helium-3 diffusion magnetic resonance imaging (HP MRI). This study aimed to characterize lung inflation over a broad range of inflation volume and pressure values in two different models of emphysema, as well as in normal lungs. Elastase-treated rats (n = 7) and healthy controls (n = 7) were imaged with HP MRI. Gradual inflation was achieved by incremental changes to both inflation volume and airway pressure. The apparent diffusion coefficient (ADC) was measured at each level of inflation and fitted to the corresponding airway pressures as the second-order response equation, with minimizing residue (χ2 < 0.001). A biphasic ADC response was detected, with an initial ADC increase followed by a decrease at airway pressures >18 cmH2O. Discrimination between treated and control rats was optimal when airway pressure was intermediate (between 10 and 11 cmH2O). Similar findings were confirmed in mice following long-term exposure to cigarette smoke, where optimal discrimination between treated and healthy mice occurred at a similar airway pressure as in the rats. We subsequently explored the evolution of ADC measured at the intermediate inflation level in mice after prolonged smoke exposure and found a significant increase (P < 0.01) in ADC over time. Our results demonstrate that measuring ADC at intermediate inflation enhances the distinction between healthy and diseased lungs, thereby establishing a model that may improve the diagnostic accuracy of future HP gas diffusion studies.


Asunto(s)
Pulmón/patología , Enfisema Pulmonar/patología , Animales , Imagen de Difusión por Resonancia Magnética/métodos , Modelos Animales de Enfermedad , Helio/química , Ratones , Ratones Endogámicos C57BL , Elastasa Pancreática/administración & dosificación , Presión , Ratas , Ratas Sprague-Dawley , Humo/efectos adversos
3.
J Pharmacol Exp Ther ; 363(1): 114-125, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28790194

RESUMEN

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key regulator of oxidative stress and cellular repair and can be activated through inhibition of its cytoplasmic repressor, Kelch-like ECH-associated protein 1 (Keap1). Several small molecule disrupters of the Nrf2-Keap1 complex have recently been tested and/or approved for human therapeutic use but lack either potency or selectivity. The main goal of our work was to develop a potent, selective activator of NRF2 as protection against oxidative stress. In human bronchial epithelial cells, our Nrf2 activator, 3-(pyridin-3-ylsulfonyl)-5-(trifluoromethyl)-2H-chromen-2-one (PSTC), induced Nrf2 nuclear translocation, Nrf2-regulated gene expression, and downstream signaling events, including induction of NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme activity and heme oxygenase-1 protein expression, in an Nrf2-dependent manner. As a marker of subsequent functional activity, PSTC restored oxidant (tert-butyl hydroperoxide)-induced glutathione depletion. The compound's engagement of the Nrf2 signaling pathway translated to an in vivo setting, with induction of Nrf2-regulated gene expression and NQO1 enzyme activity, as well as restoration of oxidant (ozone)-induced glutathione depletion, occurring in the lungs of PSTC-treated rodents. Under disease conditions, PSTC engaged its target, inducing the expression of Nrf2-regulated genes in human bronchial epithelial cells derived from patients with chronic obstructive pulmonary disease, as well as in the lungs of cigarette smoke-exposed mice. Subsequent to the latter, a dose-dependent inhibition of cigarette smoke-induced pulmonary inflammation was observed. Finally, in contrast with bardoxolone methyl and sulforaphane, PSTC did not inhibit interleukin-1ß-induced nuclear factor-κB translocation or insulin-induced S6 phosphorylation in human cells, emphasizing the on-target activity of this compound. In summary, we characterize a potent, selective Nrf2 activator that offers protection against pulmonary oxidative stress in several cellular and in vivo models.


Asunto(s)
Cumarinas/uso terapéutico , Células Epiteliales/efectos de los fármacos , Pulmón/efectos de los fármacos , Factor 2 Relacionado con NF-E2/agonistas , Estrés Oxidativo/efectos de los fármacos , Neumonía/prevención & control , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Sulfonas/uso terapéutico , Animales , Western Blotting , Línea Celular , Núcleo Celular/metabolismo , Cumarinas/administración & dosificación , Cumarinas/sangre , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Células Epiteliales/metabolismo , Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Células HEK293 , Humanos , Pulmón/metabolismo , Ratones Endogámicos C57BL , NAD(P)H Deshidrogenasa (Quinona)/genética , Factor 2 Relacionado con NF-E2/genética , Ozono/toxicidad , Neumonía/etiología , Neumonía/metabolismo , Transporte de Proteínas , ARN Interferente Pequeño/genética , Ratas Wistar , Fumar/efectos adversos , Sulfonas/administración & dosificación , Sulfonas/sangre , Transfección
4.
Mutagenesis ; 32(3): 343-353, 2017 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-27993944

RESUMEN

Inhalation of airborne toxicants such as cigarette smoke and ozone is a shared health risk among the world's populations. The use of toxic herbicides like paraquat (PQ) is restricted by many countries, yet in the developing world PQ has demonstrable ill effects. The present study examined changes in pulmonary function, mitochondrial DNA (mtDNA) integrity and markers of DNA repair induced by acute or repeated exposure of PQ to rats. Similar to cigarette smoke and ozone, PQ promotes oxidative stress, and the impact of PQ on mtDNA was compared with that obtained with these agents. Tracheal instillation (i.t.) of PQ (0.01-0.075 mg/kg) dose dependently increased Penh (dyspnoea) by 48 h while body weight and temperature declined. Lung wet weight and the wet/dry weight ratio rose; for the latter, by as much as 52%. At low doses (0.02 and 0.03 mg/kg), PQ increased Penh by about 7.5-fold at 72 h. It quickly waned to near baseline levels. The lung wet/dry weight ratio remained elevated 7 days after administration coincident with marked inflammatory cell infiltrate. Repeated administration of PQ (1 per week for 8 weeks) resulted in a similar rise in Penh on the first instillation, but the magnitude of this response was markedly attenuated upon subsequent exposures. Pulmonary [lactate] and catalase activity, [8-oxodG] and histone fragmentation (cell death) were significantly increased. Repeated PQ instillation downregulated the expression of the mitochondrial-encoded genes, mtATP8, mtNd2 and mtcyB and nuclear ones for the DNA glycosylases, Ogg1, Neil1, Neil2 and Neil3. Ogg1 protein content decreased after acute and repeated PQ administration. mtDNA damage or changes in mtDNA copy number were evident in lungs of PQ-, cigarette smoke- and ozone-exposed animals. Taken together, these data indicate that loss of pulmonary function and inflammation are coupled to the loss of mtDNA integrity and DNA repair capability following exposure to airborne toxicants.


Asunto(s)
Daño del ADN , ADN Glicosilasas/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , ADN Mitocondrial/efectos de los fármacos , Pulmón/efectos de los fármacos , Paraquat/toxicidad , 8-Hidroxi-2'-Desoxicoguanosina , Animales , ADN Glicosilasas/genética , ADN Mitocondrial/metabolismo , Desoxiguanosina/análogos & derivados , Regulación hacia Abajo , Femenino , Herbicidas/administración & dosificación , Herbicidas/farmacología , Herbicidas/toxicidad , Instilación de Medicamentos , Pulmón/metabolismo , Pulmón/fisiopatología , Masculino , Ratones , Estrés Oxidativo , Paraquat/administración & dosificación , Paraquat/farmacología , Ratas , Tráquea
5.
J Immunol ; 195(10): 4841-52, 2015 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-26438525

RESUMEN

By congenic strain mapping using autoimmune NOD.C57BL/6J congenic mice, we demonstrated previously that the type 1 diabetes (T1D) protection associated with the insulin-dependent diabetes (Idd)10 locus on chromosome 3, originally identified by linkage analysis, was in fact due to three closely linked Idd loci: Idd10, Idd18.1, and Idd18.3. In this study, we define two additional Idd loci--Idd18.2 and Idd18.4--within the boundaries of this cluster of disease-associated genes. Idd18.2 is 1.31 Mb and contains 18 genes, including Ptpn22, which encodes a phosphatase that negatively regulates T and B cell signaling. The human ortholog of Ptpn22, PTPN22, is associated with numerous autoimmune diseases, including T1D. We, therefore, assessed Ptpn22 as a candidate for Idd18.2; resequencing of the NOD Ptpn22 allele revealed 183 single nucleotide polymorphisms with the C57BL/6J (B6) allele--6 exonic and 177 intronic. Functional studies showed higher expression of full-length Ptpn22 RNA and protein, and decreased TCR signaling in congenic strains with B6-derived Idd18.2 susceptibility alleles. The 953-kb Idd18.4 locus contains eight genes, including the candidate Cd2. The CD2 pathway is associated with the human autoimmune disease, multiple sclerosis, and mice with NOD-derived susceptibility alleles at Idd18.4 have lower CD2 expression on B cells. Furthermore, we observed that susceptibility alleles at Idd18.2 can mask the protection provided by Idd10/Cd101 or Idd18.1/Vav3 and Idd18.3. In summary, we describe two new T1D loci, Idd18.2 and Idd18.4, candidate genes within each region, and demonstrate the complex nature of genetic interactions underlying the development of T1D in the NOD mouse model.


Asunto(s)
Antígenos CD2/genética , Cromosomas de los Mamíferos/genética , Diabetes Mellitus Tipo 1/genética , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genética , Alelos , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Antígenos CD2/inmunología , Cromosomas de los Mamíferos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Regulación de la Expresión Génica/inmunología , Sitios Genéticos/inmunología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Datos de Secuencia Molecular , Proteína Tirosina Fosfatasa no Receptora Tipo 22/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T/inmunología , Linfocitos T/patología
6.
Toxicol Pathol ; 43(8): 1162-5, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26353977

RESUMEN

Rodent lungs are routinely examined after intratracheal instillation (IT) of fixative. This study compares the histopathologic appearance of the lung after IT fixation with air inflation (AI) followed by immersion fixation. Lungs from mice chronically exposed to cigarette smoke were fixed either by IT or by AI. Increased numbers of macrophages with differing distributions were seen in both groups. Lungs fixed by IT had prominent, large macrophages floating in the alveolar lumina, as well as macrophage clusters and loose aggregates, often near terminal airways. Macrophages in lungs fixed by AI were randomly distributed throughout the lung, lying singly along alveolar walls, with large numbers visible in the interstitium. Clusters of macrophages were seen in the airways after AI but were fewer after IT fixation. The effects of intratracheal fixation on lung macrophages need to be considered carefully when assessing the significance of changes in macrophage appearance and distribution.


Asunto(s)
Pulmón/citología , Pulmón/patología , Macrófagos Alveolares/citología , Macrófagos Alveolares/patología , Fijación del Tejido/métodos , Fijación del Tejido/normas , Animales , Modelos Animales de Enfermedad , Femenino , Pulmón/química , Pulmón/efectos de los fármacos , Macrófagos Alveolares/química , Macrófagos Alveolares/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Humo/efectos adversos , Nicotiana
7.
Am J Physiol Lung Cell Mol Physiol ; 304(5): L312-23, 2013 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-23292810

RESUMEN

The role of T cells in chronic obstructive pulmonary disease (COPD) is not well understood. We have previously demonstrated that chronic cigarette smoke exposure can lead to the accumulation of CD4(+) and CD8(+) T cells in the alveolar airspaces in a mouse model of COPD, implicating these cells in disease pathogenesis. However, whether specific inhibition of T cell responses represents a therapeutic strategy has not been fully investigated. In this study inhibition of T cell responses through specific depleting antibodies, or the T cell immunosuppressant drug cyclosporin A, prevented airspace enlargement and neutrophil infiltration in a mouse model of chronic cigarette smoke exposure. Furthermore, individual inhibition of either CD4(+) T helper or CD8(+) T cytotoxic cells prevented airspace enlargement to a similar degree, implicating both T cell subsets as critical mediators of the adaptive immune response induced by cigarette smoke exposure. Importantly, T cell depletion resulted in significantly decreased levels of the Th17-associated cytokine IL-17A, and of caspase 3 and caspase 7 gene expression and activity, induced by cigarette smoke exposure. Finally, inhibition of T cell responses in a therapeutic manner also inhibited cigarette smoke-induced airspace enlargement, IL-17A expression, and neutrophil influx in mice. Together these data demonstrate for the first time that therapeutic inhibition of T cell responses may be efficacious in the treatment of COPD. Given that broad immunosuppression may be undesirable in COPD patients, this study provides proof-of-concept for more targeted approaches to inhibiting the role of T cells in emphysema development.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Alveolos Pulmonares/patología , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Fumar , Animales , Caspasa 3/sangre , Caspasa 7/biosíntesis , Caspasa 7/genética , Ciclosporina , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Terapia de Inmunosupresión , Interleucina-17/sangre , Mediciones del Volumen Pulmonar , Activación de Linfocitos , Depleción Linfocítica , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila/inmunología , Alveolos Pulmonares/inmunología , Enfermedad Pulmonar Obstructiva Crónica/patología , Contaminación por Humo de Tabaco
8.
Prostaglandins Other Lipid Mediat ; 104-105: 25-31, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23434473

RESUMEN

Soluble epoxide hydrolase (sEH, EPHX2) metabolizes eicosanoid epoxides, including epoxyeicosatrienoic acids (EETs) to the corresponding dihydroxyeicosatrienoic acids (DHETs), and leukotoxin (LTX) to leukotoxin diol (LTX diol). EETs, endothelium-derived hyperpolarizing factors, exhibit potentially beneficial properties, including anti-inflammatory effects and vasodilation. A novel, potent, selective inhibitor of recombinant human, rat and mouse sEH, GSK2256294A, exhibited potent cell-based activity, a concentration-dependent inhibition of the conversion of 14,15-EET to 14,15-DHET in human, rat and mouse whole blood in vitro, and a dose-dependent increase in the LTX/LTX diol ratio in rat plasma following oral administration. Mice receiving 10 days of cigarette smoke exposure concomitant with oral administration of GSK2256294A exhibited significant, dose-dependent reductions in pulmonary leukocytes and keratinocyte chemoattractant (KC, CXCL1) levels. Mice receiving oral administration of GSK2256294A following 10 days of cigarette smoke exposure exhibited significant reductions in pulmonary leukocytes compared to vehicle-treated mice. These data indicate that GSK2256294A attenuates cigarette smoke-induced inflammation by both inhibiting its initiation and/or maintenance and promoting its resolution. Collectively, these data indicate that GSK2256294A would be an appropriate agent to evaluate the role of sEH in clinical studies, for example in diseases where cigarette smoke is a risk factor, such as chronic obstructive pulmonary disease (COPD) and cardiovascular disease.


Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Ciclohexilaminas/farmacología , Epóxido Hidrolasas/antagonistas & inhibidores , Leucocitos/efectos de los fármacos , Pulmón/efectos de los fármacos , Triazinas/farmacología , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Administración Oral , Adulto , Animales , Quimiocina CXCL1/biosíntesis , Relación Dosis-Respuesta a Droga , Epóxido Hidrolasas/metabolismo , Exotoxinas/metabolismo , Femenino , Humanos , Inflamación/enzimología , Inflamación/etiología , Inflamación/patología , Inflamación/prevención & control , Recuento de Leucocitos , Leucocitos/metabolismo , Leucocitos/patología , Pulmón/enzimología , Pulmón/patología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Estrés Oxidativo/efectos de los fármacos , Ratas , Ácidos Esteáricos/metabolismo , Contaminación por Humo de Tabaco/efectos adversos
9.
Bioorg Med Chem Lett ; 21(15): 4409-15, 2011 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-21733692

RESUMEN

A series of azepanone inhibitors of cathepsin S is described. Selectivity over both cathepsin K and cathepsin L was achieved by varying the P2 substituent. Ultimately, a balanced potency and selectivity profile was achieved in compound 39 possessing a 1-methylcyclohexyl alanine at P2 and nicotinamide as the P' substituent. The cellular potency of selected analogs is also described.


Asunto(s)
Azepinas/química , Catepsinas/antagonistas & inhibidores , Niacinamida/análogos & derivados , Inhibidores de Proteasas/química , Alanina/química , Azepinas/síntesis química , Azepinas/farmacología , Sitios de Unión , Catepsina K/antagonistas & inhibidores , Catepsina K/metabolismo , Catepsina L/antagonistas & inhibidores , Catepsina L/metabolismo , Catepsinas/metabolismo , Simulación por Computador , Humanos , Niacinamida/síntesis química , Niacinamida/química , Niacinamida/farmacología , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Estereoisomerismo , Relación Estructura-Actividad
10.
J Pharmacol Exp Ther ; 330(3): 922-31, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19498103

RESUMEN

Clinical utility of phosphodiesterase 4 (PDE4) inhibitors as anti-inflammatory agents has, to date, been limited by adverse effects including nausea and emesis, making accurate assessment of emetic versus anti-inflammatory potencies critical to the development of inhibitors with improved therapeutic indices. In the present study we determined the in vitro and in vivo anti-inflammatory potencies of the first-generation PDE4 inhibitor, rolipram, the second-generation inhibitors, roflumilast and cilomilast, and a novel third generation inhibitor, 1-ethyl-5-{5-[(4-methyl-1-piperazinyl)methyl]-1,3,4-oxadiazol-2-yl}-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyridin-4-amine (EPPA-1). The rank-order potency against lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha production by human peripheral blood mononuclear cells was roflumilast (IC(50) = 5 nM) > EPPA-1 (38) > rolipram (269) > cilomilast (389), and against LPS-induced pulmonary neutrophilia in the rat was EPPA-1 (D(50) = 0.042 mg/kg) > roflumilast (0.24) > rolipram (3.34) > cilomilast (4.54). Pica, the consumption of non-nutritive substances in response to gastrointestinal stress, was used as a surrogate measure for emesis, giving a rank-order potency of rolipram (D(50) = 0.495 mg/kg) > roflumilast (1.6) > cilomilast (6.4) > EPPA-1 (24.3). The low and high emetogenic activities of EPPA-1 and rolipram, respectively, detected in the pica model were confirmed in a second surrogate model of emesis, reversal of alpha(2)-adrenoceptor-mediated anesthesia in the mouse. The rank order of therapeutic indices derived in the rat [(pica D(50))/(neutrophilia D(50))] was EPPA-1 (578) > roflumilast (6.4) > cilomilast (1.4) > rolipram (0.15), consistent with the rank order derived in the ferret [(emesis D(50))/(neutrophilia D(50))]. These data validate rat pica feeding as a surrogate for PDE4 inhibitor-induced emesis in higher species, and identify EPPA-1 as a novel PDE4 inhibitor with an improved therapeutic index.


Asunto(s)
Inhibidores de Fosfodiesterasa 4 , Inhibidores de Fosfodiesterasa/farmacología , Pica/psicología , Piperazinas/farmacología , Piridinas/farmacología , Vómitos/inducido químicamente , Aminopiridinas/farmacología , Animales , Benzamidas/farmacología , Ácidos Carboxílicos/farmacología , Ácidos Ciclohexanocarboxílicos , Ciclopropanos/farmacología , Hurones , Humanos , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos C57BL , Neutrófilos/efectos de los fármacos , Nitrilos/farmacología , Pica/inducido químicamente , Ratas , Ratas Endogámicas Lew , Receptores Adrenérgicos alfa 2/efectos de los fármacos , Receptores Adrenérgicos alfa 2/fisiología , Rolipram/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores
11.
Immunol Lett ; 121(1): 13-21, 2008 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-18706444

RESUMEN

Recently, patients with tobacco smoke induced emphysema have been shown to exhibit classical signs of T cell mediated autoimmunity characterized by autoantibody production and Th1 type responses. As the recently described Th17 type subset has been found to play a role in the pathogenesis of a number of autoimmune diseases previously considered to be Th1 driven, we sought to examine whether a Th17 type response was associated with airspace enlargement in a murine model of emphysema. Six to eight months exposure of mice to inhalation of mainstream cigarette smoke led to progressive airspace enlargement as defined by morphometric analysis. Flow cytometric analysis of the bronchoalveolar lavage (BAL) from these mice demonstrated a significant increase in the overall number of both CD4+ and CD8+ T cells present. These cells were subsequently examined for skewing towards a Th1, Th2 or Th17 phenotype by intracellular cytokine analysis. Distinct populations of BAL CD4+ T cells were found to express IFN-gamma or IL-17 demonstrating the presence of both a Th1 and Th17 type response. No expression of the Th2 associated cytokine IL-4 was detected. Further analysis of this Th17 subset demonstrated that the majority of cells with this effector phenotype express the chemokine receptor CCR6. Together these data identify a novel T cell subset associated with pulmonary inflammation as a result of cigarette smoke exposure. Given the reported roles of CCR6 and IL-17 in promoting pulmonary inflammation, this subset may play an important role in the pathogenesis of cigarette smoke induced autoimmunity.


Asunto(s)
Interleucina-17/inmunología , Pulmón/inmunología , Neumonía/inmunología , Enfisema Pulmonar/inmunología , Fumar/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Autoinmunidad , Líquido del Lavado Bronquioalveolar/inmunología , Modelos Animales de Enfermedad , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Interleucina-4/inmunología , Interleucina-4/metabolismo , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Neumonía/metabolismo , Neumonía/patología , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patología , Receptores CCR6/inmunología , Receptores CCR6/metabolismo , Fumar/patología , Subgrupos de Linfocitos T/metabolismo
12.
J Clin Invest ; 128(6): 2281-2296, 2018 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-29533925

RESUMEN

Recent studies reveal that airway epithelial cells are critical pulmonary circadian pacemaker cells, mediating rhythmic inflammatory responses. Using mouse models, we now identify the rhythmic circadian repressor REV-ERBα as essential to the mechanism coupling the pulmonary clock to innate immunity, involving both myeloid and bronchial epithelial cells in temporal gating and determining amplitude of response to inhaled endotoxin. Dual mutation of REV-ERBα and its paralog REV-ERBß in bronchial epithelia further augmented inflammatory responses and chemokine activation, but also initiated a basal inflammatory state, revealing a critical homeostatic role for REV-ERB proteins in the suppression of the endogenous proinflammatory mechanism in unchallenged cells. However, REV-ERBα plays the dominant role, as deletion of REV-ERBß alone had no impact on inflammatory responses. In turn, inflammatory challenges cause striking changes in stability and degradation of REV-ERBα protein, driven by SUMOylation and ubiquitination. We developed a novel selective oxazole-based inverse agonist of REV-ERB, which protects REV-ERBα protein from degradation, and used this to reveal how proinflammatory cytokines trigger rapid degradation of REV-ERBα in the elaboration of an inflammatory response. Thus, dynamic changes in stability of REV-ERBα protein couple the core clock to innate immunity.


Asunto(s)
Relojes Circadianos/inmunología , Ritmo Circadiano/inmunología , Homeostasis/inmunología , Inmunidad Innata , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/inmunología , Neumonía/inmunología , Animales , Relojes Circadianos/genética , Ritmo Circadiano/genética , Homeostasis/genética , Ratones , Ratones Transgénicos , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/genética , Neumonía/genética , Neumonía/patología , Proteolisis , Sumoilación/genética , Sumoilación/inmunología
13.
J Med Chem ; 59(8): 3991-4006, 2016 04 28.
Artículo en Inglés | MEDLINE | ID: mdl-27031670

RESUMEN

KEAP1 is the key regulator of the NRF2-mediated cytoprotective response, and increasingly recognized as a target for diseases involving oxidative stress. Pharmacological intervention has focused on molecules that decrease NRF2-ubiquitination through covalent modification of KEAP1 cysteine residues, but such electrophilic compounds lack selectivity and may be associated with off-target toxicity. We report here the first use of a fragment-based approach to directly target the KEAP1 Kelch-NRF2 interaction. X-ray crystallographic screening identified three distinct "hot-spots" for fragment binding within the NRF2 binding pocket of KEAP1, allowing progression of a weak fragment hit to molecules with nanomolar affinity for KEAP1 while maintaining drug-like properties. This work resulted in a promising lead compound which exhibits tight and selective binding to KEAP1, and activates the NRF2 antioxidant response in cellular and in vivo models, thereby providing a high quality chemical probe to explore the therapeutic potential of disrupting the KEAP1-NRF2 interaction.


Asunto(s)
Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Células Cultivadas , Cristalografía por Rayos X , Descubrimiento de Drogas , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/química , Ratones , Factor 2 Relacionado con NF-E2/química , Unión Proteica
14.
J Appl Physiol (1985) ; 112(1): 135-48, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21940853

RESUMEN

Changes in lung function and structure were studied using hyperpolarized (3)He MRI in an elastase-induced murine model of emphysema. The combined analysis of the apparent diffusion coefficient (ADC) and fractional ventilation (R) were used to distinguish emphysematous changes and also to develop a model for classifying sections of the lung into diseased and normal. Twelve healthy male BALB/c mice (26 ± 2 g) were randomized into healthy and elastase-induced mice and studied ∼8-11 wk after model induction. ADC and R were measured at a submillimeter planar resolution. Chord length (L(x)) data were analyzed from histology samples from the corresponding imaged slices. Logistic regression was applied to estimate the probability that an imaged pixel came from a diseased animal, and bootstrap methods (1,000 samples) were used to compare the regression results for the morphological and imaging results. Multivariate ANOVA (MANOVA) was used to analyze transformed ADC (ADC(BC)), and R (R(BC)) data and also to control for the experiment-wide error rate. MANOVA and ANOVA showed that elastase induced a statistically measureable change in the average transformed L(x) and ADC(BC) but not in the average R(BC). Marginal mean analysis demonstrated that ADC(BC) was on average 0.19 [95% confidence interval (CI): 0.16, 0.22] higher in the emphysema group, whereas R(BC) was on average 0.05 (95% CI: 0.04, 0.06) lower. Logistic regression supported the hypothesis that ADC(BC) and R(BC), together, were better at differentiating normal from diseased tissue than either measurement alone. The odds ratios for ADC(BC) and R(BC) were 7.73 (95% CI: 5.23, 11.42) and 9.14 × 10(-5) (95% CI: 3.33 × 10(-5), 25.06 × 10(-5)), respectively. Using a 50% probability cutoff, this model classified 70.6% of pixels correctly. The sensitivity and specificity of this model at the 50% cutoff were 74.9% and 65.2%, respectively. The area under the receiver operating characteristic curve was 0.76 (95% CI: 0.74, 0.78). The regression model presented can be used to map MRI data to disease probability maps. These probability maps present a future possibility of using both measurements in a more clinically feasible method of diagnosing this disease.


Asunto(s)
Modelos Animales de Enfermedad , Pulmón/patología , Pulmón/fisiología , Elastasa Pancreática/toxicidad , Enfisema Pulmonar/patología , Enfisema Pulmonar/fisiopatología , Análisis de Varianza , Animales , Modelos Logísticos , Masculino , Ratones , Ratones Endogámicos BALB C , Enfisema Pulmonar/inducido químicamente , Distribución Aleatoria
15.
J Immunol ; 180(12): 7989-8003, 2008 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-18523262

RESUMEN

Members of the papain family of cysteine proteases (cathepsins) mediate late stage processing of MHC class II-bound invariant chain (Ii), enabling dissociation of Ii, and binding of antigenic peptide to class II molecules. Recognition of cell surface class II/Ag complexes by CD4(+) T cells then leads to T cell activation. Herein, we demonstrate that a pan-active cathepsin inhibitor, SB-331750, attenuated the processing of whole cell Ii p10 to CLIP by Raji cells, and DBA/1, SJL/J, and C57BL/6 splenocytes. In Raji cells and C57BL/6 splenocytes, SB-331750 inhibited class II-associated Ii processing and reduced surface class II/CLIP expression, whereas in SB-331750-treated DBA/1 and SJL/J splenocytes, class II-associated Ii processing intermediates were undetectable. Incubation of lymph node cells/splenocytes from collagen-primed DBA/1 mice and myelin basic protein-primed SJL/J mice with Ag in the presence of SB-331750 resulted in concentration-dependent inhibition of Ag-induced proliferation. In vivo administration of SB-331750 to DBA/1, SJL/J, and C57BL/6 mice inhibited splenocyte processing of whole cell Ii p10 to CLIP. Prophylactic administration of SB-331750 to collagen-immunized/boosted DBA/1 mice delayed the onset and reduced the severity of collagen-induced arthritis (CIA), and reduced paw tissue levels of IL-1beta and TNF-alpha. Similarly, treatment of myelin basic protein-primed SJL/J lymph node cells with SB-331750 delayed the onset and reduced the severity of adoptively transferred experimental autoimmune encephalomyelitis (EAE). Therapeutic administration of SB-331750 reduced the severity of mild/moderate CIA and EAE. These results indicate that pharmacological inhibition of cathepsins attenuates CIA and EAE, potentially via inhibition of Ii processing, and subsequent Ag-induced T cell activation.


Asunto(s)
Antígenos de Diferenciación de Linfocitos B/metabolismo , Artritis Experimental/prevención & control , Azepinas/administración & dosificación , Benzofuranos/administración & dosificación , Catepsinas/antagonistas & inhibidores , Colágeno Tipo II/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Leucina/análogos & derivados , Activación de Linfocitos/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Piridinas/administración & dosificación , Animales , Artritis Experimental/enzimología , Artritis Experimental/inmunología , Azepinas/uso terapéutico , Benzofuranos/uso terapéutico , Bovinos , Línea Celular Tumoral , Células Cultivadas , Inhibidores de Cisteína Proteinasa/administración & dosificación , Inhibidores de Cisteína Proteinasa/uso terapéutico , Encefalomielitis Autoinmune Experimental/enzimología , Femenino , Humanos , Leucina/administración & dosificación , Leucina/uso terapéutico , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Procesamiento Proteico-Postraduccional/inmunología , Piridinas/uso terapéutico , Bazo/citología , Bazo/efectos de los fármacos , Bazo/enzimología
16.
J Pharmacol Exp Ther ; 312(1): 373-81, 2005 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-15316093

RESUMEN

Demonstration that IkappaB kinase 2 (IKK-2) plays a pivotal role in the nuclear factor-kappaB-regulated production of proinflammatory molecules by stimuli such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 suggests that inhibition of IKK-2 may be beneficial in the treatment of rheumatoid arthritis. In the present study, we demonstrate that a novel, potent (IC(50) = 17.9 nM), and selective inhibitor of human IKK-2, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1), inhibits lipopolysaccharide-induced human monocyte production of TNF-alpha, IL-6, and IL-8 with an IC(50) = 170 to 320 nM. Prophylactic administration of TPCA-1 at 3, 10, or 20 mg/kg, i.p., b.i.d., resulted in a dose-dependent reduction in the severity of murine collagen-induced arthritis (CIA). The significantly reduced disease severity and delay of disease onset resulting from administration of TPCA-1 at 10 mg/kg, i.p., b.i.d. were comparable to the effects of the antirheumatic drug, etanercept, when administered prophylactically at 4 mg/kg, i.p., every other day. Nuclear localization of p65, as well as levels of IL-1beta, IL-6, TNF-alpha, and interferon-gamma, were significantly reduced in the paw tissue of TPCA-1- and etanercept-treated mice. In addition, administration of TPCA-1 in vivo resulted in significantly decreased collagen-induced T cell proliferation ex vivo. Therapeutic administration of TPCA-1 at 20 mg/kg, but not at 3 or 10 mg/kg, i.p., b.i.d., significantly reduced the severity of CIA, as did etanercept administration at 12.5 mg/kg, i.p., every other day. These results suggest that reduction of proinflammatory mediators and inhibition of antigen-induced T cell proliferation are mechanisms underlying the attenuation of CIA by the IKK-2 inhibitor, TPCA-1.


Asunto(s)
Amidas/uso terapéutico , Antiasmáticos/uso terapéutico , Artritis Experimental/tratamiento farmacológico , Citocinas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Tiofenos/uso terapéutico , Adenosina Trifosfato/metabolismo , Amidas/farmacología , Animales , Antiasmáticos/farmacología , Artritis Experimental/inducido químicamente , Artritis Experimental/metabolismo , Artritis Experimental/prevención & control , Unión Competitiva , Proliferación Celular/efectos de los fármacos , Quimiocinas/metabolismo , Colágeno , Citocinas/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Quinasa I-kappa B , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos DBA , Monocitos/efectos de los fármacos , Monocitos/metabolismo , FN-kappa B/metabolismo , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Tiofenos/farmacología , Factor de Transcripción ReIA , Factor de Necrosis Tumoral alfa/metabolismo
17.
J Immunol ; 169(11): 6435-44, 2002 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-12444152

RESUMEN

Much evidence implicates IL-8 as a major mediator of inflammation and joint destruction in rheumatoid arthritis. The effects of IL-8 and its related ligands are mediated via two receptors, CXCR1 and CXCR2. In the present study, we demonstrate that a potent and selective nonpeptide antagonist of human CXCR2 potently inhibits (125)I-labeled human IL-8 binding to, and human IL-8-induced calcium mobilization mediated by, rabbit CXCR2 (IC(50) = 40.5 and 7.7 nM, respectively), but not rabbit CXCR1 (IC(50) = >1000 and 2200 nM, respectively). These data suggest that the rabbit is an appropriate species in which to examine the anti-inflammatory effects of a human CXCR2-selective antagonist. In two acute models of arthritis in the rabbit induced by knee joint injection of human IL-8 or LPS, and a chronic Ag (OVA)-induced arthritis model, administration of the antagonist at 25 mg/kg by mouth twice a day significantly reduced synovial fluid neutrophils, monocytes, and lymphocytes. In addition, in the more robust LPS- and OVA-induced arthritis models, which were characterized by increased levels of proinflammatory mediators in the synovial fluid, TNF-alpha, IL-8, PGE(2), leukotriene B(4), and leukotriene C(4) levels were significantly reduced, as was erythrocyte sedimentation rate, possibly as a result of the observed decreases in serum TNF-alpha and IL-8 levels. In vitro, the antagonist potently inhibited human IL-8-induced chemotaxis of rabbit neutrophils (IC(50) = 0.75 nM), suggesting that inhibition of leukocyte migration into the knee joint is a likely mechanism by which the CXCR2 antagonist modulates disease.


Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Artritis Experimental/inmunología , Artritis Experimental/prevención & control , Receptores de Interleucina-8B/antagonistas & inhibidores , Urea/farmacología , Enfermedad Aguda , Animales , Artritis Experimental/etiología , Artritis Reumatoide/etiología , Artritis Reumatoide/inmunología , Quimiotaxis de Leucocito/efectos de los fármacos , Enfermedad Crónica , Femenino , Humanos , Técnicas In Vitro , Interleucina-8/administración & dosificación , Interleucina-8/inmunología , Interleucina-8/metabolismo , Lipopolisacáridos/toxicidad , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Conejos , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Urea/análogos & derivados
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