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ACS Med Chem Lett ; 6(8): 845-9, 2015 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-26288682


Early hit to lead work on a pyrrolopyridine chemotype provided access to compounds with biochemical and cellular potency against Janus kinase 2 (JAK2). Structure-based drug design along the extended hinge region of JAK2 led to the identification of an important H-bond interaction with the side chain of Tyr 931, which improved JAK family selectivity. The 4,5-dimethyl thiazole analogue 18 demonstrated high levels of JAK family selectivity and was identified as a promising lead for the program.

J Biol Chem ; 287(44): 37406-19, 2012 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-22948158


The innate immune system protects the host from bacterial and viral invasion. Surfactant protein A (SPA), a lung-specific collectin, stimulates macrophage chemotaxis. However, the mechanisms regulating this function are unknown. Hyaluronan (HA) and its receptors RHAMM (receptor for HA-mediated motility, CD168) and CD44 also regulate cell migration and inflammation. We therefore examined the role of HA, RHAMM, and CD44 in SPA-stimulated macrophage chemotaxis. Using antibody blockade and murine macrophages, SPA-stimulated macrophage chemotaxis was dependent on TLR2 but not the other SPA receptors examined. Anti-TLR2 blocked SPA-induced production of TGFß. In turn, TGFß1-stimulated chemotaxis was inhibited by HA-binding peptide and anti-RHAMM antibody but not anti-TLR2 antibody. Macrophages from TLR2(-/-) mice failed to migrate in response to SPA but responded normally to TGFß1 and HA, effects that were blocked by anti-RHAMM antibody. Macrophages from WT and CD44(-/-) mice had similar responses to SPA, whereas those from RHAMM(-/-) mice had decreased chemotaxis to SPA, TGFß1, and HA. In primary macrophages, SPA-stimulated TGFß production was dependent on TLR2, JNK, and ERK but not p38. Pam3Cys, a specific TLR2 agonist, stimulated phosphorylation of JNK, ERK, and p38, but only JNK and ERK inhibition blocked Pam3Cys-stimulated chemotaxis. We have uncovered a novel pathway for SPA-stimulated macrophage chemotaxis where SPA stimulation via TLR2 drives JNK- and ERK-dependent TGFß production. TGFß1, in turn, stimulates macrophage chemotaxis in a RHAMM and HA-dependent manner. These findings are highly relevant to the regulation of innate immune responses by SPA with key roles for specific components of the extracellular matrix.

Quimiotaxia , Proteínas da Matriz Extracelular/metabolismo , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/fisiologia , Macrófagos/fisiologia , Proteína A Associada a Surfactante Pulmonar/fisiologia , Receptor 2 Toll-Like/metabolismo , Fator de Crescimento Transformador beta1/fisiologia , Animais , Linhagem Celular , Citoesqueleto/metabolismo , Proteínas da Matriz Extracelular/genética , Técnicas de Inativação de Genes , Receptores de Hialuronatos/genética , Ácido Hialurônico/metabolismo , Lipoproteínas/farmacologia , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , Camundongos , Vison , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Pseudópodes/metabolismo , Pseudópodes/fisiologia , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/genética , Fator de Crescimento Transformador beta1/metabolismo
Pediatrics ; 120(2): 346-53, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17671061


OBJECTIVES: We hypothesized that inhaled nitric oxide treatment of premature infants at risk for bronchopulmonary dysplasia would not adversely affect endogenous surfactant function or composition. METHODS: As part of the Nitric Oxide Chronic Lung Disease Trial of inhaled nitric oxide, we examined surfactant in a subpopulation of enrolled infants. Tracheal aspirate fluid was collected at specified intervals from 99 infants with birth weights <1250 g who received inhaled nitric oxide (20 ppm, weaned to 2 ppm) or placebo gas for 24 days. Large-aggregate surfactant was analyzed for surface activity with a pulsating bubble surfactometer and for surfactant protein contents with an immunoassay. RESULTS: At baseline, before administration of study gas, surfactant function and composition were comparable in the 2 groups, and there was a positive correlation between minimum surface tension and severity of lung disease for all infants. Over the first 4 days of treatment, minimum surface tension increased in placebo-treated infants and decreased in inhaled nitric oxide-treated infants. There were no significant differences between groups in recovery of large-aggregate surfactant or contents of surfactant protein A, surfactant protein B, surfactant protein C, or total protein, normalized to phospholipid. CONCLUSIONS: We conclude that inhaled nitric oxide treatment for premature infants at risk of bronchopulmonary dysplasia does not alter surfactant recovery or protein composition and may improve surfactant function transiently.

Recém-Nascido Prematuro/fisiologia , Óxido Nítrico/administração & dosagem , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/fisiologia , Proteínas Associadas a Surfactantes Pulmonares/química , Proteínas Associadas a Surfactantes Pulmonares/fisiologia , Administração por Inalação , Displasia Broncopulmonar/tratamento farmacológico , Displasia Broncopulmonar/fisiopatologia , Displasia Broncopulmonar/prevenção & controle , Feminino , Humanos , Recém-Nascido , Masculino , Alvéolos Pulmonares/química , Tensão Superficial/efeitos dos fármacos
Am J Physiol Lung Cell Mol Physiol ; 292(1): L249-57, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16997883


In the fetal lung, endogenous transforming growth factor (TGF)-beta inhibits early morphogenesis and blocks hormone-induced type II cell differentiation. We hypothesized that endogenous TGF-beta inhibits type II cell differentiation and that the stimulatory effects of glucocorticoids result in part from suppression of TGF-beta. Epithelial cells were isolated from human fetal lung and cultured under defined conditions with and without dexamethasone plus cAMP to promote type II cell differentiation. Control cells produced TGF-beta, which was activated in part by alpha(V)beta(6)-integrin. Treatment with dexamethasone, but not cAMP, reduced TGF-beta1 and -beta2 transcripts and TGF-beta bioactivity in culture medium. To examine the effects of decreased TGF-beta in the absence of glucocorticoid, cells were treated with antibodies to TGF-beta and its receptors. By real-time RT-PCR, antibody blockade of TGF-beta reduced serpine1, a TGF-beta-inducible gene, and increased gene expression for sftpa, sftpb, sftpc, and titf1, mimicking the response to hormone treatment. By microarray analysis, 29 additional genes were induced by both TGF-beta antibody and hormone treatment, and 20 other genes were repressed by both treatments. For some genes, the fold response was comparable for antibody and hormone treatment. We conclude that endogenous TGF-beta suppresses expression of surfactant proteins and selected other type II cell genes in fetal lung, in part secondary to increased expression of titf1, and we propose that the mechanism of glucocorticoid-induced type II cell differentiation includes antagonism of TGF-beta gene suppression. Surfactant production during fetal development is likely influenced by relative levels of TGF-beta and glucocorticoids.

Glucocorticoides/farmacologia , Pulmão/citologia , Pulmão/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Dexametasona/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feto/citologia , Feto/metabolismo , Perfilação da Expressão Gênica , Humanos , Pulmão/metabolismo , Vison , Proteínas Associadas a Surfactantes Pulmonares/genética , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Transdução de Sinais , Fatores de Transcrição , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/genética
J Nutr ; 135(2): 160-4, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15671207


Carotenoids, plant pigments with potent antioxidant activity, are implicated in chronic disease protection. They are absorbed from the diet and transported by plasma lipoproteins. Monocytes, as circulating blood cells, are exposed to carotenoid-rich lipoproteins. Such exposure may lead to enrichment with carotenoids and may affect the functions of monocyte-derived macrophages. This study explored the effect of cellular enrichment in vitro with beta-carotene, lycopene, or lutein on monocyte/macrophage function, using U937 cells as a model. Cell proliferation, production of reactive oxygen species, and cell-substrate adhesion were examined. Maximal carotenoid levels in medium supplemented with preenriched human serum were 2-8 mumol/L; incubation for 1-6 d resulted in 0.2-1.1 nmol carotenoid/mg cell protein (0.25-1 nmol/10(6) cells), approximately 10-fold more than that reported in normal tissue in vivo but within the range that might be anticipated with dietary supplementation. beta-Carotene, lycopene, and lutein markedly inhibited the proliferation of U937 cells, to an extent similar to or greater than that due to phorbol myristic acetate, a known differentiation/activation agent. Lycopene, but not beta-carotene or lutein, caused a significant increase in reactive oxygen species, indicating the induction of cell differentiation. Adhesion and LDL oxidation were unaffected. Thus, cellular carotenoids inhibit proliferation, and for lycopene at least, this may involve cell differentiation. The effectiveness of lycopene, a nonprovitamin A carotenoid, is consistent with a vitamin A-independent pathway modulating cell function.

Carotenoides/sangue , Carotenoides/farmacologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Macrófagos/citologia , Monócitos/citologia , Linhagem Celular , Humanos , Cinética , Luteína/farmacologia , Licopeno , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Acetato de Tetradecanoilforbol , Células U937 , beta Caroteno/farmacologia