Efeitos da melatonina na inflamação de vias aéreas em modelos experimentais de asma, DPOC e de sobreposição Asma-DPOC

Background and Objectives: According to previous studies, melatonin, an indolamine, may possibly reduce dyspnea in patients with asthma or COPD. Additionally, it has also been proven, through experimental studies, that the molecule possibly plays an anti-inflammatory role, because it might inhibit transcription of NFk-B in experimental models of asthma and COPD. This study aims to investigate the effects of melatonin in experimental models of asthma, COPD and Asthma-COPD Overlap (OCP). Methods: 64 mice were divided into 8 groups in order to induce COPD (group “ALS”), asthma (“OVA”) or OC (“ACO”). The control group (“SAL”) received saline. The treatment groups (“+MEL”) were submitted to both disease protocols and also received Melatonin (intraperitoneally). After the protocols, the exhaled nitric oxide (Eno) and the total and differential cells of the bronchoalveolar lavage fluid were evaluated. Results: OVA+MEL (11.3±1.65ppb) showed reduction in Eno compared to OVA (24.17±4.30ppb). In the analysis of the bronchoalveolar lavage fluid cells, OVA+MEL (11.3±1.65x104 cells/mL) and ACO+MEL (2.17±0.63x104 cells/mL) showed a reduction in the amount of total cells compared to OVA (25.70±4.59x104 cells/mL) and ACO (14.33±3.11x104 cells/mL), respectively. There was a decrease in eosinophil count in OVA+MEL (5.37±1.41x104 cells/mL) and ACO+MEL (0.87±0.36x104 cells/mL) compared to OVA (18.67±4.01x104 cells/mL) and ACO (1.45±0.41x104 cells/mL), respectively. Additionally, the number of lymphocytes decreased in OVA + MEL (1.00±0.24x104 céls./mL) compared to OVA (3.94±1.15x104 cells/mL). The number of macrophages also decreased in OVA + MEL (3.09±0.39x104 cells/mL) and ACO + MEL (1.10±0.27x104 cells/mL) compared to OVA (7.26±1.93x104 cells/mL) and ACO (6.41±1.54x104 cells/mL), respectively. There was no difference in the comparison of neutrophil counts in the different groups analyzed.Justificativa e Objetivos: De acordo com estudos anteriores, a melatonina, uma indolamina, pode possivelmente reduzir a dispneia em pacientes que apresentam asma ou DPOC. Adicionalmente, foi provado igualmente, através dos estudos experimentais, que a molécula joga possivelmente um papel antiinflamatório, porque pôde inibir a transcrição de NFk-B em modelos experimentais da asma e do COPD. Este estudo tem como objetivo investigar os efeitos da melatonina em modelos experimentais de asma, DPOC e Sobreposição da Asma-DPOC (ACO). Métodos: 64 camundongos foram divididos em 8 grupos, a fim de induzir DPOC (grupo “ELA”), asma (“OVA”) ou ACO (“ACO”). O grupo controle (“SAL”) recebeu solução salina. Os grupos de tratamento (“+MEL”) foram submetidos aos dois protocolos de doença e também receberam Melatonina (intraperitonealmente). Após os protocolos, foram avaliados o óxido nítrico exalado (Eno), bem como as células totais e diferenciais do fluido do lavado broncoalveolar. Resultados: OVA+MEL (11.3±1.65ppb) apresentou redução em eNO em comparação a OVA (24.17±4.30ppb). Além disso, OVA+MEL (11.3±1.65x104 céls./mL) e ACO+MEL (2.17±0.63x104 céls./mL) apresentaram redução na quantidade de células totais em comparação a OVA (25.70±4.59x104 céls./mL) e ACO (14.33±3.11x104 céls./mL), respectivamente. Houve queda na contagem de eosinófilos em OVA+MEL (5.37±1.41x104 céls./mL) e em ACO+MEL (0.87±0.36x104 céls./mL) em comparação a OVA (18.67±4.01x104 céls./mL) e ACO (1.45±0.41x104 céls./mL), respectivamente. Adicionalmente, o número de linfócitos apresentou redução em OVA+MEL (1.00±0.24x104 céls./mL) em comparação a OVA (3.94±1.15x104 céls./mL). O número de macrófagos também apresentou redução em OVA+MEL (3.09±0.39x104 céls./mL) e em ACO+MEL (1.10±0.27x104 céls./mL) em comparação a OVA (7.26±1.93x104 céls./mL) e ACO (6.41±1.54x104 céls./mL), respectivamente (p<0.05 para todas as comparações). Não houve diferença na comparação da contagem de neutrófilos nos diferentes grupos analisados. Conclusão: O tratamento com melatonina mostrou-se, em modelo experimental de asma e ACO, efetivo na redução de parâmetros pró-inflamatórios, podendo representar um papel importante no controle de tais patologias. Além disso, o tratamento com melatonina em modelos experimentais de lesão pulmonar induzida por elastase não se mostrou eficaz no controle de parâmetros pró-inflamatórios no fluido do lavado broncoalveolar, sendo necessários mais estudos para o entendimento dos mecanismos de ação da melatonina no tecido pulmonar e nas vias aéreas

‘‘Shadow’’ OSCE examiner. A cross-sectional study comparing the ‘‘shadow’’ examiner with the original OSCE examiner format

OSCEOBJECTIVES: Feedback is a powerful learning tool, but a lack of appropriate feedback is a very common complaint from learners to teachers. To improve opportunities for feedback on objective structured clinical examinations (OSCEs), a modified examiner role, termed the ‘‘shadow’’ examiner, was tested. This study aims to present and analyze comparisons between the ‘‘shadow’’ examiner and the original OSCE examiner format. METHODS: In 2011, experiments were carried out with modifications to the examiner’s role to define the ‘‘shadow’’ examiner format. From February 2012 to May 2014, research was conducted with 415 6th-year medical students. Of these students, 316 were randomly assigned to assessments by both ‘‘shadow’’ and ‘‘fixed’’ examiners. Pearson correlation analysis with linear regression, Student’s t-tests and Bland-Altman plots were the statistical methods used to compare the assessment modes. To strengthen the analysis, checklist items were classified by domain. RESULTS: High correlations between the ‘‘shadow’’ and ‘‘fixed’’ examiners’ global scores were observed. The results of the analysis of specific domains demonstrated higher correlations for cognitive scores and lower correlations for affective scores. No statistically significant differences between the mean examiner global scores were found. The Bland-Altman analysis showed that the ‘‘shadow’’ examiners’ affective scores were significantly higher than those of the ‘‘fixed’’ examiners, but the magnitude of this difference was small. CONCLUSION: The modified examiner role did not lead to any important bias in the students’ scores compared with the original OSCE examiner format. This new strategy may provide important insights for formative assessments of clinical performance

Biseugenol Exhibited Anti-Inflammatory and Anti-Asthmatic Effects in an Asthma Mouse Model of Mixed-Granulocytic Asthma

In the present work, the anti-inflammatory and antiasthmatic potential of biseugenol, isolated as the main component from n-hexane extract from leaves of Nectandra leucantha and chemically prepared using oxidative coupling from eugenol, was evaluated in an experimental model of mixed-granulocytic asthma. Initially, in silico studies of biseugenol showed good predictions for drug-likeness, with adherence to Lipinski’s rules of five (RO5), good Absorption, Distribution, Metabolism and Excretion (ADME) properties and no alerts for Pan-Assay Interference Compounds (PAINS), indicating adequate adherence to perform in vivo assays. Biseugenol (20 mg·kg−1) was thus administered intraperitoneally (four days of treatment) and resulted in a significant reduction in both eosinophils and neutrophils of bronchoalveolar lavage fluid in ovalbumin-sensitized mice with no statistical difference from dexamethasone (5 mg·kg−1). As for lung function parameters, biseugenol (20 mg·kg−1) significantly reduced airway and tissue damping in comparison to ovalbumin group, with similar efficacy to positive control dexamethasone. Airway hyperresponsiveness to intravenous methacholine was reduced with biseugenol but was inferior to dexamethasone in higher doses. In conclusion, biseugenol displayed antiasthmatic effects, as observed through the reduction of inflammation and airway hyperresponsiveness, with similar effects to dexamethasone, on mixed-granulocytic ovalbumin-sensitized miceFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP: 2018/06088-

Effects of Anti-IL-17 on Inflammation, Remodeling, and Oxidative Stress in an Experimental Model of Asthma Exacerbated by LPS

Inflammation plays a central role in the development of asthma, which is considered an allergic disease with a classic Th2 inflammatory profile. However, cytokine IL-17 has been examined to better understand the pathophysiology of this disease. Severe asthmatic patients experience frequent exacerbations, leading to infection, and subsequently show altered levels of inflammation that are unlikely to be due to the Th2 immune response alone. This study estimates the effects of anti-IL-17 therapy in the pulmonary parenchyma in a murine asthma model exacerbated by LPS. BALB/c mice were sensitized with intraperitoneal ovalbumin and repeatedly exposed to inhalation with ovalbumin, followed by treatment with or without anti-IL-17. Twenty-four hours prior to the end of the 29-day experimental protocol, the two groups received LPS (0.1 mg/ml intratracheal OVA-LPS and OVA-LPS IL-17). We subsequently evaluated bronchoalveolar lavage fluid, performed a lung tissue morphometric analysis, and measured IL-6 gene expression. OVA-LPS-treated animals treated with anti-IL-17 showed decreased pulmonary inflammation, edema, oxidative stress, and extracellular matrix remodeling compared to the non-treated OVA and OVA-LPS groups (p < 0.05). The anti-IL-17 treatment also decreased the numbers of dendritic cells, FOXP3, NF-kappa B, and Rho kinase 1-and 2-positive cells compared to the non-treated OVA and OVA-LPS groups (p < 0.05). In conclusion, these data suggest that inhibition of IL-17 is a promising therapeutic avenue, even in exacerbated asthmatic patients, and significantly contributes to the control of Th1/Th2/Th17 inflammation, chemokine expression, extracellular matrix remodeling, and oxidative stress in a murine experimental asthma model exacerbated by LPS.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)National Council of Scientific and the Technological Development (CNPq)Laboratory of Medical InvestigationsUniv Sao Paulo, Sch Med, Dept Med Sci, Sao Paulo, Brazil|Hosp Sirio Libanes, Sao Paulo, BrazilUniv Fed Sao Paulo, Inst Biomed Sci, Dept Biol Sci, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biol Sci, Sao Paulo, BrazilUniv Fed Sao Paulo, Inst Biomed Sci, Dept Biol Sci, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biol Sci, Sao Paulo, BrazilFAPESP: 2013/17944-1Laboratory of Medical Investigations: LIM-20 FMUSPWeb of Scienc

Modulation of the oscillatory mechanics of lung tissue and the oxidative stress response induced by arginase inhibition in a chronic allergic inflammation model

Abstract\ud \ud \ud \ud Background\ud The importance of the lung parenchyma in the pathophysiology of asthma has previously been demonstrated. Considering that nitric oxide synthases (NOS) and arginases compete for the same substrate, it is worthwhile to elucidate the effects of complex NOS-arginase dysfunction in the pathophysiology of asthma, particularly, related to distal lung tissue. We evaluated the effects of arginase and iNOS inhibition on distal lung mechanics and oxidative stress pathway activation in a model of chronic pulmonary allergic inflammation in guinea pigs.\ud \ud \ud \ud Methods\ud Guinea pigs were exposed to repeated ovalbumin inhalations (twice a week for 4 weeks). The animals received 1400 W (an iNOS-specific inhibitor) for 4 days beginning at the last inhalation. Afterwards, the animals were anesthetized and exsanguinated; then, a slice of the distal lung was evaluated by oscillatory mechanics, and an arginase inhibitor (nor-NOHA) or vehicle was infused in a Krebs solution bath. Tissue resistance (Rt) and elastance (Et) were assessed before and after ovalbumin challenge (0.1%), and lung strips were submitted to histopathological studies.\ud \ud \ud \ud Results\ud Ovalbumin-exposed animals presented an increase in the maximal Rt and Et responses after antigen challenge (p<0.001), in the number of iNOS positive cells (p<0.001) and in the expression of arginase 2, 8-isoprostane and NF-kB (p<0.001) in distal lung tissue. The 1400 W administration reduced all these responses (p<0.001) in alveolar septa. Ovalbumin-exposed animals that received nor-NOHA had a reduction of Rt, Et after antigen challenge, iNOS positive cells and 8-isoprostane and NF-kB (p<0.001) in lung tissue. The activity of arginase 2 was reduced only in the groups treated with nor-NOHA (p <0.05). There was a reduction of 8-isoprostane expression in OVA-NOR-W compared to OVA-NOR (p<0.001).\ud \ud \ud \ud Conclusions\ud In this experimental model, increased arginase content and iNOS-positive cells were associated with the constriction of distal lung parenchyma. This functional alteration may be due to a high expression of 8-isoprostane, which had a procontractile effect. The mechanism involved in this response is likely related to the modulation of NF-kB expression, which contributed to the activation of the arginase and iNOS pathways. The association of both inhibitors potentiated the reduction of 8-isoprostane expression in this animal model.FAPESP and LIM20HCFMUSP.FAPESP and LIM-20-HC-FMUSP

Y-27632 is associated with corticosteroid-potentiated control of pulmonary remodeling and inflammation in guinea pigs with chronic allergic inflammation

Abstract\ud \ud Background\ud Previously, we showed that treatment with the Rho-kinase inhibitor Y-27632 was able to control airway responsiveness, inflammation, remodeling, and oxidative stress in an animal model of asthma, suggesting that this drug is beneficial in asthma. However, studies evaluating the effects of these inhibitors in conjunction with corticosteroids on chronic pulmonary inflammation have not been conducted. Therefore, we evaluated the effects of treatment with the Rho-kinase inhibitor Y-27632, with or without concurrent dexamethasone treatment, on airway and lung tissue mechanical responses, inflammation, extracellular matrix remodeling, and oxidative stress in guinea pigs with chronic allergic inflammation.\ud \ud \ud Methods\ud The guinea pigs were subjected to seven ovalbumin or saline inhalation exposures. Treatment with Y-27632 (1 mM) and dexamethasone (2 mg/kg) started at the fifth inhalation. Seventy-two hours after the seventh inhalation, the pulmonary mechanics were evaluated and exhaled nitric oxide (ENO) levels were determined. The lungs were removed and histological analysis was performed using morphometry.\ud \ud \ud Results\ud The treatment of guinea pigs with the Rho-kinase inhibitor and dexamethasone (ORC group) decreased ENO, the maximal mechanical responses after antigen challenge, inflammation, extracellular matrix remodeling and oxidative stress in the lungs.\ud This therapeutic strategy reduced the levels of collagen and IFN-γ in the airway walls, as well as IL-2, IFN-γ, 8-iso-PGF2α and NF-κB in the distal parenchyma, when compared to isolated treatment with corticosteroid or Rho-kinase inhibitor (P < 0.05) and reduced the number of TIMP-1-positive cells and eosinophils in the alveolar septa compared to corticosteroid-treated animals (P < 0.05). The combined treatment with the Rho-kinase inhibitor and the corticosteroid provided maximal control over the remodeling response and inflammation in the airways and parenchyma.\ud \ud \ud Conclusions\ud Rho-kinase inhibition, alone or in combination with corticosteroids, can be considered a future pharmacological tool for the control of asthma.We thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for their financial support

Protective Effects of Anti-IL17 on Acute Lung Injury Induced by LPS in Mice

Introduction: T helper 17 (Th17) has been implicated in a variety of inflammatory lung and immune system diseases. However, little is known about the expression and biological role of IL-17 in acute lung injury (ALI). We investigated the mechanisms involved in the effect of anti-IL17 in a model of lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice.Methods: Mice were pre-treated with anti-IL17, 1h before saline/LPS intratracheal administration alongside non-treated controls and levels of exhaled nitric oxide (eNO), cytokine expression, extracellular matrix remodeling and oxidative stress, as well as immune cell counts in bronchoalveolar lavage fluid (BALF), and respiratory mechanics were assessed in lung tissue.Results: LPS instillation led to an increase in multiple cytokines, proteases, nuclear factor-κB, and Forkhead box P3 (FOXP3), eNO and regulators of the actomyosin cytoskeleton, the number of CD4+ and iNOS-positive cells as well as the number of neutrophils and macrophages in BALF, resistance and elastance of the respiratory system, ARG-1 gene expression, collagen fibers, and actin and 8-iso-PGF2α volume fractions. Pre-treatment with anti-IL17 led to a significant reduction in the level of all assessed factors.Conclusions: Anti-IL17 can protect the lungs from the inflammatory effects of LPS-induced ALI, primarily mediated by the reduced expression of cytokines and oxidative stress. This suggests that further studies using anti-IL17 in a treatment regime would be highly worthwhile