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The Nod-like Receptor (NLR) apoptosis inhibitory proteins (NAIPs) are cytosolic receptors that sense cytosolic bacterial proteins. NAIP ligation induces its association with NLRC4, leading to the assembly of the NAIP/NLRC4 inflammasome, which induces the activation of the caspase-1 protease. Caspase-1 then cleaves pro-interleukin (IL)-1ß, pro-IL-18, and gasdermin D and induces a form of pro-inflammatory cell death, pyroptosis. These processes culminate in host defense against bacterial infection. Here we describe methods for activating NAIP/NLRC4 inflammasome signalling in human and murine macrophages and quantifying inflammasome-induced cell death.
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Proteínas de Ligação ao Cálcio , Inflamassomos , Animais , Camundongos , Humanos , Inflamassomos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Morte Celular , Caspases/metabolismo , Caspase 1/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismoRESUMO
OBJECTIVE: To investigate the role of PF-06650833, a highly potent and selective small-molecule inhibitor of interleukin-1-associated kinase 4 (IRAK4), in autoimmune pathophysiology in vitro, in vivo, and in the clinical setting. METHODS: Rheumatoid arthritis (RA) inflammatory pathophysiology was modeled in vitro through 1) stimulation of primary human macrophages with anti-citrullinated protein antibody immune complexes (ICs), 2) RA fibroblast-like synoviocyte (FLS) cultures stimulated with Toll-like receptor (TLR) ligands, as well as 3) additional human primary cell cocultures exposed to inflammatory stimuli. Systemic lupus erythematosus (SLE) pathophysiology was simulated in human neutrophils, dendritic cells, B cells, and peripheral blood mononuclear cells stimulated with TLR ligands and SLE patient ICs. PF-06650833 was evaluated in vivo in the rat collagen-induced arthritis (CIA) model and the mouse pristane-induced and MRL/lpr models of lupus. Finally, RNA sequencing data generated with whole blood samples from a phase I multiple-ascending-dose clinical trial of PF-06650833 were used to test in vivo human pharmacology. RESULTS: In vitro, PF-06650833 inhibited human primary cell inflammatory responses to physiologically relevant stimuli generated with RA and SLE patient plasma. In vivo, PF-06650833 reduced circulating autoantibody levels in the pristane-induced and MRL/lpr murine models of lupus and protected against CIA in rats. In a phase I clinical trial (NCT02485769), PF-06650833 demonstrated in vivo pharmacologic action pertinent to SLE by reducing whole blood interferon gene signature expression in healthy volunteers. CONCLUSION: These data demonstrate that inhibition of IRAK4 kinase activity can reduce levels of inflammation markers in humans and provide confidence in the rationale for clinical development of IRAK4 inhibitors for rheumatologic indications.
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Artrite Experimental/tratamento farmacológico , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Isoquinolinas/uso terapêutico , Lactamas/uso terapêutico , Macrófagos/efeitos dos fármacos , Doenças Reumáticas/tratamento farmacológico , Sinoviócitos/efeitos dos fármacos , Animais , Artrite Experimental/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Modelos Animais de Doenças , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Isoquinolinas/farmacologia , Lactamas/farmacologia , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Camundongos , Ratos , Doenças Reumáticas/imunologia , Sinoviócitos/imunologiaRESUMO
BACKGROUND: PF-06650833 is a potent, selective inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4). Two randomized, double-blind, sponsor-open phase 1 studies evaluated the safety, pharmacokinetics, and pharmacodynamics of single (SAD) and multiple ascending doses (MAD) of PF-06650833 immediate-release (IR) and modified-release (MR) oral formulations in healthy adult subjects. METHODS: Study 1 (NCT02224651) was a 96-day, placebo-substitution, SAD study of once-daily (QD) oral PF-06650833 IR 1 to 6000 mg and MR 30 to 300 mg in fasted and fed states. Study 2 (NCT02485769) was a 14-day, placebo-controlled, MAD study of PF-06650833 IR 25 to 750 mg twice daily, IR 1000 mg four times per day, IR 330 mg three times per day, and MR 300 mg QD. RESULTS: PF-06650833 was generally well tolerated, with no dose-limiting treatment-emergent adverse events (TEAEs) identified in either study. TEAEs were generally mild in severity, with headache, gastrointestinal disorders, and acne most commonly reported. No serious AEs or deaths were reported. A maximum tolerated dose was not established in either study. In the SAD study, food intake delayed absorption of IR 30 mg and increased total exposure by 33%. Delayed absorption was achieved with the MR formulation (Tmax of 1 h versus 8 h for IR 100 mg and MR 100 mg formulations, respectively). Food had no effect on total exposure for MR 30 mg, but reduced half-life 1.8-fold and increased Cmax by 62%. In the MAD study, accumulation ranged from 0.9-fold to 1.4-fold for AUCtau and 0.9-fold to 1.3-fold for Cmax. Less than 1% of the dose was recovered unchanged in urine for all dose groups, with renal clearance ranging from 14 to 23 mL/min for IR < 750 mg and MR 300 mg. There was a sustained decrease in serum high-sensitivity C-reactive protein for IR ≥ 250 mg and MR 300 mg. Based on the cholesterol/hydroxycholesterol ratio, no apparent CYP3A induction or inhibition was observed. CONCLUSIONS: PF-06650833, the first IRAK4 inhibitor to enter clinical development, has a favorable safety and pharmacokinetic profile and has shown evidence of pharmacological effect. The data support continued evaluation in human clinical trials for the treatment of rheumatic and autoimmune diseases. TRIAL REGISTRATION: Clinicaltrials.gov, NCT02224651, registered 25 August 2014; NCT02485769, registered 30 June 2015.
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Antirreumáticos/efeitos adversos , Antirreumáticos/farmacocinética , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Isoquinolinas/efeitos adversos , Isoquinolinas/farmacocinética , Adulto , Relação Dose-Resposta a Droga , Feminino , Voluntários Saudáveis , Humanos , Lactamas , Masculino , Pessoa de Meia-IdadeRESUMO
Increased reactive oxygen species (ROS) have been implicated in the pathophysiology of chronic obstructive pulmonary disease (COPD). This study examined the effect of exogenous and endogenous oxidative stress on macrophage phagocytosis in patients with COPD.Monocyte-derived macrophages (MDMs) were generated from non-smoker, smoker and COPD subjects, differentiated in either granulocyte macrophage-colony stimulating factor (G-Mφ) or macrophage-colony stimulating factor (M-Mφ). Alveolar macrophages were isolated from lung tissue or bronchoalveolar lavage fluid. Macrophages were incubated in ±200â µM H2O2 for 24â h, then exposed to fluorescently labelled Haemophilus influenzae or Streptococcus pneumoniae for 4â h, after which phagocytosis, mitochondrial ROS (mROS) and mitochondrial membrane potential (ΔΨm) were measured.Phagocytosis of bacteria was significantly decreased in both G-Mφ and M-Mφ from COPD patients compared with from non-smoker controls. In non-smokers and smokers, bacterial phagocytosis did not alter mROS or ΔΨm; however, in COPD, phagocytosis increased early mROS and decreased ΔΨm in both G-Mφ and M-Mφ. Exogenous oxidative stress reduced phagocytosis in non-smoker and COPD alveolar macrophages and non-smoker MDMs, associated with reduced mROS production.COPD macrophages show defective phagocytosis, which is associated with altered mitochondrial function and an inability to regulate mROS production. Targeting mitochondrial dysfunction may restore the phagocytic defect in COPD.
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Macrófagos Alveolares/imunologia , Mitocôndrias/metabolismo , Fagocitose/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Idoso , Bactérias , Sobrevivência Celular , Feminino , Haemophilus influenzae , Humanos , Técnicas In Vitro , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Masculino , Potencial da Membrana Mitocondrial , Microscopia Confocal , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Streptococcus pneumoniaeRESUMO
PF-06651600 was developed as an irreversible inhibitor of JAK3 with selectivity over the other three JAK isoforms. A high level of selectivity toward JAK3 is achieved by the covalent interaction of PF-06651600 with a unique cysteine residue (Cys-909) in the catalytic domain of JAK3, which is replaced by a serine residue in the other JAK isoforms. Importantly, 10 other kinases in the kinome have a cysteine at the equivalent position of Cys-909 in JAK3. Five of those kinases belong to the TEC kinase family including BTK, BMX, ITK, RLK, and TEC and are also inhibited by PF-06651600. Preclinical data demonstrate that inhibition of the cytolytic function of CD8+ T cells and NK cells by PF-06651600 is driven by the inhibition of TEC kinases. On the basis of the underlying pathophysiology of inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, alopecia areata, and vitiligo, the dual activity of PF-06651600 toward JAK3 and the TEC kinase family may provide a beneficial inhibitory profile for therapeutic intervention.
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Janus Quinase 3/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Lectinas Tipo C/antagonistas & inibidores , Lectinas Tipo C/imunologia , CamundongosRESUMO
Atopic dermatitis (AD) is a highly prevalent, chronic inflammatory skin disease that affects children and adults. The pathophysiology of AD is complex and involves skin barrier and immune dysfunction. Many immune cytokine pathways are amplified in AD, including T helper (Th) 2, Th22, Th17 and Th1. Current treatment guidelines recommend topical medications as initial therapy; however, until recently, only two drug classes were available: topical corticosteroids (TCSs) and topical calcineurin inhibitors (TCIs). Several limitations are associated with these agents. TCSs can cause a wide range of adverse effects, including skin atrophy, telangiectasia, rosacea and acne. TCIs can cause burning and stinging, and the prescribing information lists a boxed warning for a theoretical risk of malignancy. Novel medications with new mechanisms of action are necessary to provide better long-term control of AD. Phosphodiesterase 4 (PDE4) regulates cyclic adenosine monophosphate in cells and has been shown to be involved in the pathophysiology of AD, making it an attractive therapeutic target. Several PDE4 inhibitors are in clinical development for use in the treatment of AD, including crisaborole, which recently became the first topical PDE4 inhibitor approved for treatment of mild to moderate AD. This review will further describe the pathophysiology of AD, explain the possible role of PDE4 in AD and review PDE4 inhibitors currently approved or being investigated for use in AD.
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Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/fisiologia , Dermatite Atópica/metabolismo , Dermatite Atópica/prevenção & controle , Inibidores de Fosfodiesterase/farmacologia , Acetamidas/farmacologia , Compostos de Boro/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Citocinas/metabolismo , Dermatite Atópica/fisiopatologia , Aprovação de Drogas , Humanos , Sistema Imunitário , Inflamação , Ácidos Ftálicos/farmacologia , Piridinas/farmacologia , Quinazolinas/farmacologia , Risco , Pele/patologia , Talidomida/análogos & derivados , Talidomida/farmacologiaRESUMO
Background: The CCL3L1-CCR5 signaling axis is important in a number of inflammatory responses, including macrophage function, and T-cell-dependent immune responses. Small molecule CCR5 antagonists exist, including the approved antiretroviral drug maraviroc, and therapeutic monoclonal antibodies are in development. Repositioning of drugs and targets into new disease areas can accelerate the availability of new therapies and substantially reduce costs. As it has been shown that drug targets with genetic evidence supporting their involvement in the disease are more likely to be successful in clinical development, using genetic association studies to identify new target repurposing opportunities could be fruitful. Here we investigate the potential of perturbation of the CCL3L1-CCR5 axis as treatment for respiratory disease. Europeans typically carry between 0 and 5 copies of CCL3L1 and this multi-allelic variation is not detected by widely used genome-wide single nucleotide polymorphism studies. Methods: We directly measured the complex structural variation of CCL3L1 using the Paralogue Ratio Test and imputed (with validation) CCR5del32 genotypes in 5,000 individuals from UK Biobank, selected from the extremes of the lung function distribution, and analysed DNA and RNAseq data for CCL3L1 from the 1000 Genomes Project. Results: We confirmed the gene dosage effect of CCL3L1 copy number on CCL3L1 mRNA expression levels. We found no evidence for association of CCL3L1 copy number or CCR5del32 genotype with lung function. Conclusions: These results suggest that repositioning CCR5 antagonists is unlikely to be successful for the treatment of airflow obstruction.
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RATIONALE: Previous studies have identified defects in bacterial phagocytosis by alveolar macrophages (AMs) in patients with chronic obstructive pulmonary disease (COPD), but the mechanisms and clinical consequences remain incompletely defined. OBJECTIVES: To examine the effect of COPD on AM phagocytic responses and identify the mechanisms, clinical consequences, and potential for therapeutic manipulation of these defects. METHODS: We isolated AMs and monocyte-derived macrophages (MDMs) from a cohort of patients with COPD and control subjects within the Medical Research Council COPDMAP consortium and measured phagocytosis of bacteria in relation to opsonic conditions and clinical features. MEASUREMENTS AND MAIN RESULTS: COPD AMs and MDMs have impaired phagocytosis of Streptococcus pneumoniae. COPD AMs have a selective defect in uptake of opsonized bacteria, despite the presence of antipneumococcal antibodies in BAL, not observed in MDMs or healthy donor AMs. AM defects in phagocytosis in COPD are significantly associated with exacerbation frequency, isolation of pathogenic bacteria, and health-related quality-of-life scores. Bacterial binding and initial intracellular killing of opsonized bacteria in COPD AMs was not reduced. COPD AMs have reduced transcriptional responses to opsonized bacteria, such as cellular stress responses that include transcriptional modules involving antioxidant defenses and Nrf2 (nuclear factor erythroid 2-related factor 2)-regulated genes. Agonists of the cytoprotective transcription factor Nrf2 (sulforaphane and compound 7) reverse defects in phagocytosis of S. pneumoniae and nontypeable Haemophilus influenzae by COPD AMs. CONCLUSIONS: Patients with COPD have clinically relevant defects in opsonic phagocytosis by AMs, associated with impaired transcriptional responses to cellular stress, which are reversed by therapeutic targeting with Nrf2 agonists.
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Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fagocitose/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Isotiocianatos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/fisiologia , Masculino , Pessoa de Meia-Idade , Fagocitose/fisiologia , Streptococcus pneumoniae , SulfóxidosRESUMO
BACKGROUND: Airway inflammation is a feature of many respiratory diseases and there is a need for newer, more effective anti-inflammatory compounds. The aim of this study was to develop an ex vivo human lung explant model which can be used to help study the mechanisms underlying inflammatory responses and which can provide a tool to aid drug discovery for inflammatory respiratory diseases such as asthma and COPD. METHOD: Parenchymal lung tissue from 6 individual donors was dissected and cultured with two pro-inflammatory stimuli, lipopolysaccharide (LPS) (1 µg/ml) and interleukin-1 beta (IL-1ß) (10 ng/ml) in the presence or absence of dexamethasone (1 µM). Inflammatory responses were assessed using Luminex analysis of tissue culture supernatants to measure levels of 21 chemokines, growth factors and cytokines. RESULTS: A robust and reproducible inflammatory signal was detected across all donors for 12 of the analytes measured following LPS stimulation with a modest fold increase (<2-fold) in levels of CCL22, IL-4, and IL-2; increases of 2-4-fold in levels of CXCL8, VEGF and IL-6 and increases >4-fold in CCL3, CCL4, GM-CSF, IL-10, TNF-α and IL-1ß. The inflammatory signal induced by IL-1ß stimulation was less than that observed with LPS but resulted in elevated levels of 7 analytes (CXCL8, CCL3, CCL4, GM-CSF, IL-6, IL-10 and TNF-α). The inflammatory responses induced by both stimulations was supressed by dexamethasone for the majority of analytes. CONCLUSIONS: These data provide proof of concept that this ex vivo human lung explant model is responsive to inflammatory signals and could be used to investigate the anti-inflammatory effects of existing and novel compounds. In addition this model could be used to help define the mechanisms and pathways involved in development of inflammatory airway disease. ABBREVIATIONS: COPD: Chronic Obstructive Pulmonary Disease; ICS: inhaled corticosteroids; LPS: lipopolysaccharide; IL-1ß: interleukin-1 beta; PSF: penicillin, streptomycin and fungizone.
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Through fragment-based drug design focused on engaging the active site of IRAK4 and leveraging three-dimensional topology in a ligand-efficient manner, a micromolar hit identified from a screen of a Pfizer fragment library was optimized to afford IRAK4 inhibitors with nanomolar potency in cellular assays. The medicinal chemistry effort featured the judicious placement of lipophilicity, informed by co-crystal structures with IRAK4 and optimization of ADME properties to deliver clinical candidate PF-06650833 (compound 40). This compound displays a 5-unit increase in lipophilic efficiency from the fragment hit, excellent kinase selectivity, and pharmacokinetic properties suitable for oral administration.
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Descoberta de Drogas , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Isoquinolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Relação Dose-Resposta a Droga , Humanos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Isoquinolinas/administração & dosagem , Isoquinolinas/química , Lactamas , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
Background: The association of differing genotypes with disease-related phenotypic traits offers great potential to both help identify new therapeutic targets and support stratification of patients who would gain the greatest benefit from specific drug classes. Development of low-cost genotyping and sequencing has made collecting large-scale genotyping data routine in population and therapeutic intervention studies. In addition, a range of new technologies is being used to capture numerous new and complex phenotypic descriptors. As a result, genotype and phenotype datasets have grown exponentially. Genome-wide association studies associate genotypes and phenotypes using methods such as logistic regression. As existing tools for association analysis limit the efficiency by which value can be extracted from increasing volumes of data, there is a pressing need for new software tools that can accelerate association analyses on large genotype-phenotype datasets. Results: Using open innovation (OI) and contest-based crowdsourcing, the logistic regression analysis in a leading, community-standard genetics software package (PLINK 1.07) was substantially accelerated. OI allowed us to do this in <6 months by providing rapid access to highly skilled programmers with specialized, difficult-to-find skill sets. Through a crowd-based contest a combination of computational, numeric, and algorithmic approaches was identified that accelerated the logistic regression in PLINK 1.07 by 18- to 45-fold. Combining contest-derived logistic regression code with coarse-grained parallelization, multithreading, and associated changes to data initialization code further developed through distributed innovation, we achieved an end-to-end speedup of 591-fold for a data set size of 6678 subjects by 645 863 variants, compared to PLINK 1.07's logistic regression. This represents a reduction in run time from 4.8 hours to 29 seconds. Accelerated logistic regression code developed in this project has been incorporated into the PLINK2 project. Conclusions: Using iterative competition-based OI, we have developed a new, faster implementation of logistic regression for genome-wide association studies analysis. We present lessons learned and recommendations on running a successful OI process for bioinformatics.
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Crowdsourcing , Estudo de Associação Genômica Ampla , Software , Genótipo , Humanos , Invenções , Modelos Logísticos , FenótipoRESUMO
BACKGROUND: Genome-wide association studies have identified numerous genetic regions that influence cross-sectional lung function. Longitudinal decline in lung function also includes a heritable component but the genetic determinants have yet to be defined. OBJECTIVES: We aimed to determine whether regions associated with cross-sectional lung function were also associated with longitudinal decline and to seek novel variants which influence decline. METHODS: We analysed genome-wide data from 4167 individuals from the Busselton Health Study cohort, who had undergone spirometry (12â 695 observations across eight time points). A mixed model was fitted and weighted risk scores were calculated for the joint effect of 26 known regions on baseline and longitudinal changes in FEV1 and FEV1/FVC. Potential additional regions of interest were identified and followed up in two independent cohorts. RESULTS: The 26 regions previously associated with cross-sectional lung function jointly showed a strong effect on baseline lung function (p=4.44×10-16 for FEV1/FVC) but no effect on longitudinal decline (p=0.160 for FEV1/FVC). This was replicated in an independent cohort. 39 additional regions of interest (48 variants) were identified; these associations were not replicated in two further cohorts. CONCLUSIONS: Previously identified genetic variants jointly have a strong effect on cross-sectional lung function in adults but little or no effect on the rate of decline of lung function. It is possible that they influence COPD risk through lung development. Although no genetic variants have yet been associated with lung function decline at stringent genome-wide significance, longitudinal change in lung function is heritable suggesting that there is scope for future discoveries.
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Variação Genética , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Respiração/genética , Adulto , Estudos Transversais , Progressão da Doença , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Testes de Função Respiratória , Medição de Risco , Fatores de Risco , Espirometria , Fatores de Tempo , Austrália OcidentalRESUMO
By use of a structure-based computational method for identification of structurally novel Janus kinase (JAK) inhibitors predicted to bind beyond the ATP binding site, a potent series of indazoles was identified as selective pan-JAK inhibitors with a type 1.5 binding mode. Optimization of the series for potency and increased duration of action commensurate with inhaled or topical delivery resulted in potent pan-JAK inhibitor 2 (PF-06263276), which was advanced into clinical studies.
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Anti-Inflamatórios/farmacologia , Compostos Heterocíclicos com 2 Anéis/farmacologia , Indazóis/farmacologia , Janus Quinases/antagonistas & inibidores , Pneumopatias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Dermatopatias/tratamento farmacológico , Administração Cutânea , Administração por Inalação , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/toxicidade , Sítios de Ligação , Cristalografia por Raios X , Cães , Desenho de Fármacos , Hepatócitos/metabolismo , Compostos Heterocíclicos com 2 Anéis/administração & dosagem , Compostos Heterocíclicos com 2 Anéis/síntese química , Compostos Heterocíclicos com 2 Anéis/toxicidade , Humanos , Indazóis/administração & dosagem , Indazóis/síntese química , Indazóis/toxicidade , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 3/antagonistas & inibidores , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/toxicidade , Ratos , SolubilidadeRESUMO
INTRODUCTION: Genome-Wide Association Studies have identified associations between lung function measures and Chronic Obstructive Pulmonary Disease (COPD) and chromosome region 6p21 containing the gene for the Advanced Glycation End Product Receptor (AGER, encoding RAGE). We aimed to (i) characterise RAGE expression in the lung, (ii) identify AGER transcripts, (iii) ascertain if SNP rs2070600 (Gly82Ser C/T) is associated with lung function and serum sRAGE levels and (iv) identify whether the Gly82Ser variant is functionally important in altering sRAGE levels in an airway epithelial cell model. METHODS: Immunohistochemistry was used to identify RAGE protein expression in 26 human tissues and qPCR was used to quantify AGER mRNA in lung cells. Gene expression array data was used to identify AGER expression during lung development in 38 fetal lung samples. RNA-Seq was used to identify AGER transcripts in lung cells. sRAGE levels were assessed in cells and patient serum by ELISA. BEAS2B-R1 cells were transfected to overexpress RAGE protein with either the Gly82 or Ser82 variant and sRAGE levels identified. RESULTS: Immunohistochemical assessment of 6 adult lung samples identified high RAGE expression in the alveoli of healthy adults and individuals with COPD. AGER/RAGE expression increased across developmental stages in human fetal lung at both the mRNA (38 samples) and protein levels (20 samples). Extensive AGER splicing was identified. The rs2070600T (Ser82) allele is associated with higher FEV1, FEV1/FVC and lower serum sRAGE levels in UK smokers. Using an airway epithelium model overexpressing the Gly82 or Ser82 variants we found that HMGB1 activation of the RAGE-Ser82 receptor results in lower sRAGE production. CONCLUSIONS: This study provides new information regarding the expression profile and potential role of RAGE in the human lung and shows a functional role of the Gly82Ser variant. These findings advance our understanding of the potential mechanisms underlying COPD particularly for carriers of this AGER polymorphism.
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Pulmão/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Receptor para Produtos Finais de Glicação Avançada/genética , Fumar , Alelos , Brônquios/citologia , Brônquios/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feto/metabolismo , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Plasmídeos/genética , Plasmídeos/metabolismo , Polimorfismo de Nucleotídeo Único , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Splicing de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Receptor para Produtos Finais de Glicação Avançada/sangue , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Adulto JovemRESUMO
Pulmonary inflammation and bacterial colonization are central to the pathogenesis of chronic obstructive pulmonary disease (COPD). Defects in macrophage phagocytosis of both bacteria and apoptotic cells contribute to the COPD phenotype. Small molecule inhibitors with anti-inflammatory activity against p38 mitogen activated protein kinases (MAPKs), phosphatidyl-inositol-3 kinase (PI3K) and Rho kinase (ROCK) are being investigated as novel therapeutics in COPD. Concerns exist, however, about off-target effects. We investigated the effect of p38 MAPK inhibitors (VX745 and SCIO469), specific inhibitors of PI3K α (NVS-P13K-2), δ (NVS-P13K-3) or γ (NVS-P13K-5) and a ROCK inhibitor PF4950834 on macrophage phagocytosis, early intracellular killing of bacteria and efferocytosis of apoptotic neutrophils. Alveolar macrophages (AM) obtained from broncho-alveolar lavage (BAL) or monocyte-derived macrophages (MDM) from COPD patients (GOLD stage II/III) enrolled from a well characterized clinical cohort (MRC COPD-MAP consortium) or from healthy ex-smoker controls were studied. Both COPD AM and MDM exhibited lower levels of bacterial phagocytosis (using Streptococcus pneumoniae and non-typeable Haemophilus influenzae) and efferocytosis than healthy controls. None of the inhibitors altered bacterial internalization or early intracellular bacterial killing in AM or MDM. Conversely PF4950834, but not other inhibitors, enhanced efferocytosis in COPD AM and MDM. These results suggest none of these inhibitors are likely to exacerbate phagocytosis-related defects in COPD, while confirming ROCK inhibitors can enhance efferocytosis in COPD.
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BACKGROUND: Exacerbations of asthma and COPD are triggered by rhinoviruses. Uncontrolled inflammatory pathways, pathogenic bacterial burden and impaired antiviral immunity are thought to be important factors in disease severity and duration. Macrolides including azithromycin are often used to treat the above diseases, but exhibit variable levels of efficacy. Inhaled corticosteroids are also readily used in treatment, but may lack specificity. Ideally, new treatment alternatives should suppress unwanted inflammation, but spare beneficial antiviral immunity. METHODS: In the present study, we screened 225 novel macrolides and tested them for enhanced antiviral activity against rhinovirus, as well as anti-inflammatory activity and activity against Gram-positive and Gram-negative bacteria. Primary bronchial epithelial cells were grown from 10 asthmatic individuals and the effects of macrolides on rhinovirus replication were also examined. Another 30 structurally similar macrolides were also examined. RESULTS: The oleandomycin derivative Mac5, compared with azithromycin, showed superior induction (up to 5-fold, EC50â=â5-11 µM) of rhinovirus-induced type I IFNß, type III IFNλ1 and type III IFNλ2/3 mRNA and the IFN-stimulated genes viperin and MxA, yet had no effect on IL-6 and IL-8 mRNA. Mac5 also suppressed rhinovirus replication at 48 h, proving antiviral activity. Mac5 showed antibacterial activity against Gram-positive Streptococcus pneumoniae; however, it did not have any antibacterial properties compared with azithromycin when used against Gram-negative Escherichia coli (as a model organism) and also the respiratory pathogens Pseudomonas aeruginosa and non-typeable Haemophilus influenzae. Further non-toxic Mac5 derivatives were identified with various anti-inflammatory, antiviral and antibacterial activities. CONCLUSIONS: The data support the idea that macrolides have antiviral properties through a mechanism that is yet to be ascertained. We also provide evidence that macrolides can be developed with anti-inflammatory, antibacterial and antiviral activity and show surprising versatility depending on the clinical need.
Assuntos
Antibacterianos/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Antivirais/química , Antivirais/farmacologia , Descoberta de Drogas , Interferons/imunologia , Macrolídeos/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/uso terapêutico , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/isolamento & purificação , Anti-Inflamatórios não Esteroides/uso terapêutico , Antivirais/isolamento & purificação , Antivirais/uso terapêutico , Asma/tratamento farmacológico , Brônquios/citologia , Brônquios/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Células Epiteliais/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Haemophilus influenzae/efeitos dos fármacos , Humanos , Interferon beta/imunologia , Interferons/biossíntese , Interleucina-6/imunologia , Interleucina-6/metabolismo , Interleucina-8/imunologia , Interleucina-8/metabolismo , Macrolídeos/química , Macrolídeos/uso terapêutico , Proteínas de Resistência a Myxovirus/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Proteínas/genética , Pseudomonas aeruginosa/efeitos dos fármacos , Rhinovirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Inhaled LPS causes neutrophilic airway inflammation in healthy subjects. We compared the effects of p38 MAPK inhibitors and fluticasone propionate on the LPS response. METHODS: Three randomised, double-blind, placebo-controlled, single dose crossover studies were performed. Active treatments were the oral p38 MAPK inhibitor PH-797804 30 mg (study 1), PH-797804 30 mg and the inhaled p38 MAPK inhibitor PF-03715455 20 mg (study 2) and inhaled fluticasone propionate 500 µg (study 3). The primary endpoint was sputum neutrophil percentage. RESULTS: Sputum neutrophil percentage post-LPS challenge was significantly inhibited (15.1 and 15.3% reduction) by PH-797804 compared to placebo in studies 1 and 2 (p = 0.0096 and 0.0001, respectively), and by PF-03715455 (8.0% reduction, p = 0.031); fluticasone propionate had no effect. PH-797804 significantly inhibited the increase in inflammatory mediators (IL-6, MCP-1, MIP1ß and CC16) in sputum supernatant, while PF-03715455 had no effect. PH-797804 and PF-03715455 both inhibited IL-6, MCP-1, MIP1ß, CC16 and CRP levels in plasma, with PH-797804 having greater effects. Fluticasone propionate had no effect on sputum supernatant or plasma biomarkers. CONCLUSIONS: PH-797804 had the greatest impact on neutrophilic airway inflammation. Oral administration of p38 MAPK inhibitors may optimise pulmonary anti-inflammatory effects.
Assuntos
Anti-Inflamatórios/farmacologia , Compostos Azabicíclicos/farmacologia , Benzamidas/farmacologia , Fluticasona/farmacologia , Mediadores da Inflamação/metabolismo , Compostos de Metilureia/farmacologia , Piridonas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Administração por Inalação , Adolescente , Adulto , Testes de Provocação Brônquica , Estudos Cross-Over , Método Duplo-Cego , Feminino , Humanos , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Escarro/citologia , Adulto JovemRESUMO
In chronic obstructive pulmonary disease (COPD), oxidative stress regulates the inflammatory response of bronchial epithelium and monocytes/macrophages through kinase modulation and has been linked to glucocorticoid unresponsiveness. Glycogen synthase-3ß (GSK3ß) inactivation plays a key role in mediating signaling processes upon reactive oxygen species (ROS) exposure. We hypothesized that GSK3ß is involved in oxidative stress-induced glucocorticoid insensitivity in COPD. We studied levels of phospho-GSK3ß-Ser9, a marker of GSK3ß inactivation, in lung sections and cultured monocytes and bronchial epithelial cells of COPD patients, control smokers, and nonsmokers. We observed increased levels of phospho-GSK3ß-Ser9 in monocytes, alveolar macrophages, and bronchial epithelial cells from COPD patients and control smokers compared with nonsmokers. Pharmacological inactivation of GSK3ß did not affect CXCL8 or granulocyte-macrophage colony-stimulating factor (GM-CSF) expression but resulted in glucocorticoid insensitivity in vitro in both inflammatory and structural cells. Further mechanistic studies in monocyte and bronchial epithelial cell lines showed that GSK3ß inactivation is a common effector of oxidative stress-induced activation of the MEK/ERK-1/2 and phosphatidylinositol 3-kinase/Akt signaling pathways leading to glucocorticoid unresponsiveness. In primary monocytes, the mechanism involved modulation of histone deacetylase 2 (HDAC2) activity in response to GSK3ß inactivation. In conclusion, we demonstrate for the first time that ROS-induced glucocorticoid unresponsiveness in COPD is mediated through GSK3ß, acting as a ROS-sensitive hub.
Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Quinase 3 da Glicogênio Sintase/fisiologia , Doença Pulmonar Obstrutiva Crônica/enzimologia , Idoso , Células Cultivadas , Dexametasona/uso terapêutico , Feminino , Expressão Gênica/efeitos dos fármacos , Glucocorticoides/uso terapêutico , Glicogênio Sintase Quinase 3 beta , Histona Desacetilase 2/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Macrófagos Alveolares/enzimologia , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Mucosa Respiratória/enzimologia , Transdução de SinaisRESUMO
BACKGROUND: CD8+ T-cells are located in the small airways of COPD patients and may contribute to pathophysiology. CD8+ cells express the chemokine receptor, CXCR3 that binds CXCL9, CXCL10 and CXCL11, which are elevated in the airways of COPD patients. These chemokines are released from airway epithelial cells via activation of receptor associated Janus kinases (JAK). This study compared the efficacy of two structurally dissimilar pan-JAK inhibitors, PF956980 and PF1367550, and the glucocorticosteroid dexamethasone, in BEAS-2B and human primary airway epithelial cells from COPD patients and control subjects. METHODS: Cells were stimulated with either IFNγ alone or with TNFα, and release of CXCL9, CXCL10 and CXCL11 measured by ELISA and expression of CXCL9, CXCL10 and CXCL11 by qPCR. Activation of JAK signalling was assessed by STAT1 phosphorylation and DNA binding. RESULTS: There were no differences in the levels of release of CXCL9, CXCL10 and CXCL11 from primary airway epithelial cells from any of the subjects or following stimulation with either IFNγ alone or with TNFα. Dexamethasone did not inhibit CXCR3 chemokine release from stimulated BEAS-2B or primary airway epithelial cells. However, both JAK inhibitors suppressed this response with PF1367550 being ~50-65-fold more potent than PF956980. The response of cells from COPD patients did not differ from controls with similar responses regardless of whether inhibitors were added prophylactically or concomitant with stimuli. These effects were mediated by JAK inhibition as both compounds suppressed STAT1 phosphorylation and DNA-binding of STAT1 and gene transcription. CONCLUSIONS: These data suggest that the novel JAK inhibitor, PF1367550, is more potent than PF956980 and that JAK pathway inhibition in airway epithelium could provide an alternative anti-inflammatory approach for glucocorticosteroid-resistant diseases including COPD.