Thesis (MS)--Montana State University--Bozeman, 2008. / Typescript. Chairperson, Graduate Committee: Mary P. Miles. Includes bibliographical references (leaves 64-67).

Diss. (sammanfattning) Göteborg : Göteborgs universitet , 2008. / Härtill 5 uppsatser.

Made available in DSpace on 2015-05-14T12:59:34Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2299557 bytes, checksum: 01deb75646894a343a19a53f79cf2519 (MD5) Previous issue date: 2012-02-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Ouabain (OUA), a potent inhibitor of the Na+,K+-ATPase pump, was identified as an endogenous substance of human plasma. In recent years, ouabain was shown to affect various immunological processes. Mechanisms that involve cellular differentiation, proliferation, activation and migration, as well as inflammatory mediators release, are activated during inflammation and homeostasis is usually reestablished. This study demonstrated the modulatory ability of OUA on inflammatory process. Aim: This study aimed to evaluate ouabain immunomodulatory role on acute inflammatory process using a murine model. Methods: Initially, a dose and time-response curve was performed with OUA (0.10 mg/kg, 0.31 mg/kg and 0.56 mg/kg) intraperitoneally administered on the paw edema induced by zymosan (10 mg/mL). Mice were also intraperitoneally (i.p.) stimulated with zymosan (2 mg/mL). After 4h, the peritoneal fluid was removed for total and differential cell counts. Neutrophils and macrophages population, as well as cell viability, were analyzed using an annexin KIT by flow cytometry. The concentrations of the cytokines IL-1β, TNF-α, IL-6 and IL-10 in peritoneal lavage fluids were assayed using ELISA kit. Ouabain influence in the vascular permeability increase was determined using evans blue dye. OUA, in vitro, influence on nitric oxide (NO) production was also studied. Results: It was observed that OUA 0,56 mg/kg injected for three consecutive days prevented zymosan edema formation . After induction of inflammation, treatment with OUA led to a 42% reduction in the total cell numbers in the peritoneal cavity, as a reflex of the inhibition of polymorphonuclear leukocytes (54%), which was not due to cell apoptosis. Ouabain also decreased zymosan-induced plasma exudation (33%). Furthermore, OUA decreased the levels of TNF-α (64%) and IL-1β (63%), without interference on IL-6 and IL-10 levels. It was also demonstrated, using peritoneal macrophages, that ouabain did not interfere on LPS induced NO production. Conclusion: Ouabain modulated the acute inflammatory response induced by zymosan. However, further studies are necessary to elucidate the mechanisms involved / A ouabaína (OUA), um potente inibidor da Na+/K+-ATPase, foi identificada como uma substância endógena presente no plasma humano. Nos últimos anos, foi evidenciado que a OUA é capaz de interferir em diversos aspectos do sistema imunológico. Durante o processo inflamatório, são ativados mecanismos que envolvem a diferenciação, proliferação, ativação e migração celular, além da liberação de mediadores inflamatórios, e geralmente, ocorre o retorno à homeostasia. Este trabalho demonstrou a capacidade moduladora da OUA no processo inflamatório. Objetivo: Avaliar o papel imunomodulador da OUA na inflamação aguda em modelo murino. Métodos: Inicialmente, foi realizada uma curva de tempo e dose-resposta com a OUA (0,10 mg/kg; 0,31 mg/kg e 0,56 mg/kg) administrada de forma intra-peritoneal (i.p.) no modelo de edema de pata induzido por zimosan (10mg/mL). Os camundongos também foram estimulados com zimosan i.p.(2mg/ml). Após 4h, o fluido peritoneal foi removido para a contagem total e diferencial das células. Foi realizada a análise das populações de neutrófilos e macrófagos, além da viabilidade celular utilizando o kit anexina por citometria de fluxo. As concentrações das citocinas IL-1β, TNF-α, IL-6 e IL-10 no fluido peritoneal foram testadas por ELISA. Foi determinada a interferência do tratamento com a OUA no aumento da permeabilidade vascular. Também foi estudado, in vitro, o efeito de diferentes concentrações de OUA (10 nM, 100 nM e 1000 nM) na produção de óxido nítrico (NO). Resultados: No modelo do edema de pata, observamos que são necessários três dias consecutivos de tratamento na dose de 0,56 mg/kg para que a atividade anti-inflamatória da OUA seja identificada. No modelo de peritonite induzida por zimosan, o pré-tratamento com a OUA reduziu em 42% no número total de células na cavidade peritoneal, como um reflexo da inibição de leucócitos polimorfonucleares (54%). No entanto, este fenômeno não esta associado a apoptose destas células. A OUA também diminuiu o extravasamento de plasma induzido por zimosan (33%) e reduziu os níveis de TNF-α (64%) e IL-1β (63%), sem alterar os níveis de IL-6 e IL-10. Na cultura de macrófagos peritoneais, a OUA não interferiu na produção de NO. Conclusão: Este conjunto de dados sugere que a OUA possui uma atividade anti-inflamatória. No entanto, mais estudos são necessários para elucidar os mecanismos envolvidos.

In cystic fibrosis (CF) therapy, the recent approval of CF-transmembrane conductance regulator (CFTR) channel modulators is considered to be the major breakthrough. However, the current first-line approach based mainly on pulmonary function to measure effects of the novel therapy, tested by forced expiratory volumes in one second (FEV1), provides restricted sensitivity to detect early structural damages. Accordingly, there is a need for new sensitive surrogate parameters. Most interestingly, these should quantify inflammation that precedes a decline of pulmonary function. We present a novel method assessing inflammatory markers in the upper airways’ epithelial lining fluid (ELF) obtained by nasal lavage (NL). In contrast to broncho-alveolar lavage, ELF sampling by NL is an attractive method due to its limited invasiveness which allows repeated analyses, even performed in a home-based setting. In a longitudinal cohort study (ClinicalTrials.gov, Identifier: NCT02311140), we assessed changes of inflammatory mediators in 259 serially obtained nasal lavages taken up to every second day before and during therapy with ivacaftor from ten CF patients carrying a G551D mutation. Patients were trained to sample NL-fluid at home, to immediately freeze and transfer chilled secretions to centers. Neutrophil Elastase, Interleukins IL-1b, IL-6 and IL-8 in NL were quantified. During 8-12 weeks of ivacaftor-treatment, median values of IL-1b and IL-6 significantly declined 2.29-fold (2.97!1.30 pg/mL), and 1.13-fold (6.48!5.72 pg/mL), respectively. In parallel, sweat tests and pulmonary function improved considerably. This is the first study assessing changes of airway inflammation on a day-to-day basis in CF patients receiving a newly administered CFTR-modulator therapy. It proves a decline of airway inflammation during ivacaftor-therapy.