Recruitment and derecruitment (R/D) of airways is known to significantly influence mechanical properties of the respiratory system during artificial ventilation, particularly in states of lung injury. The prevailing view of this phenomenon treats airway R/D as a static function of pressure. Recent experimental and clinical data suggests that this is not the case, but rather that R/D is an inherently dynamic process. In order to quantitatively assess the dynamics of lung recruitment during mechanical ventilation we extended a mathematical model by Bates and Irvin (9) for the purpose of fitting experimental data. The model of the lung consists of a parallel network of flow pathways with identical resistive and elastic elements. Each pathway is allowed to be either open, whereby it accumulates flow and decreases overall lung stiffness, or closed, increasing lung elastance and not participating in ventilation. The pathways are characterized by unique critical closing and opening pressures, and opening and closing velocities, each chosen from probability distribution functions. The rate of transition between an open and closed state depends on the magnitude difference between the pressure in the respiratory system and each unit’s critical pressure times the airway’s opening or closing velocity constant. Since the exact form of the pressure dependence governing recruitment and derecruitment remains unknown we explored four model variants to predict how opening or closing behavior is altered in injury. The lung model was coupled with a computational model of a mechanical ventilator in order to simulate elastance changes following deep inflation (DI) at three levels of Positive End Expiratory Pressure (PEEP). Elastance measurements came from healthy or lung injured mice at 4, 14, 24 or 48 hours following intratracheal instillation of saline (control) or hydrochloric acid (injury). The Nelder and Mead simplex optimization method was used to minimize error between model variants and average experimental elastance for each condition. By comparing the residual error of the fits for each model, we have demonstrated that only one variant was able to recreate both the transient response to deep inflations and the response to static PEEP. In fitting the best model to data from individual mice we obtained estimates for parameters governing opening and closing behavior. Statistics and model sensitivity were determined for each parameter in every experimental condition. Comparison of parameter values between groups revealed a significant increase in closing and opening pressures from health to injury, which worsened with increasing injury severity. The progressive increase in critical pressures as injury worsens implicates surfactant deactivation as the likely cause of increased propensity for airway closing during acute lung injury.

Background: Acute lung injury (ALI) and nosocomial pneumonia are major causes of morbidity and mortality. There are 200,000 cases per year of ALI in the US with a mortality of 40%. On the intensive care unit (ICU), ALI accounts for over 40% of all ventilated patients at any one time. Despite this huge burden on healthcare and the relatively high prevalence, no therapies currently exist in clinical practice that attenuate the condition. The pathophysiology and aetiology of ALI is multifactorial but neutrophilic influx and consequent damage to the endothelial-epithelial interface are regarded as central features. Alongside neutrophils, peripheral blood monocytes (PBMs) are recruited to the acutely inflamed lung. The role played by PBMs in perpetuating the pathogenic neutrophilic influx remains poorly characterised. Nosocomial pneumonia is also a major problem with drug resistant organisms. With the increasing prevalence of antibiotic resistance and the paucity of novel antimicrobials being generated by pharmaceutical companies, there is real concern that the end of the ‘antibiotic era’ may be approaching. AIMS 1) To develop murine models of lung inflammation and infection 2) To establish the role of the PBM in perpetuating the neutrophilic response in ALI 3) To develop non-invasive methodologies to study the trafficking of cells and molecular events within the inflamed lung 4) To apply a novel antimicrobial to prevent and treat nosocomial pneumonia Methods: A murine model of ALI was utilised using direct intratracheal instillation of lipopolysaccharide. To this model 3 different PBM depletion strategies were applied to study the effect on neutrophil recruitment and consequent lung injury. Non invasive optical imaging was utilised to study the effect of PBM depletion on proteolytic events within the murine lung. To understand cellular trafficking, cell labeling strategies were compared for primary murine macrophages with whole body optical imaging in mice. Murine models of Staphylococcus aureus, Pseudomonas aeruginosa and Burkholderia cepacia were established and a novel antimicrobial agent called the nonalysine like peptoid (NLLP) tested in vitro and in vivo for efficacy. Results: PBM depletion significantly attenuated neutrophil recruitment in an established model of ALI. Near infrared (NIR) optical imaging permitted the non invasive tracking of primary murine cells. A non toxic peptidomimetic agent (NLLP) possessed antimicrobial activity against gram positive and gram negative pathogens with therapeutic and prophylactic efficacy in vivo. Conclusions: PBM depletion is a potential therapeutic strategy for treating ALI. Further studies are required to determine the exact mechanism by which PBMs orchestrate neutrophil recruitment. Optical imaging is a versatile platform for molecular imaging. A novel antimicrobial agent termed NLLP has been discovered with therapeutic and prophylactic efficacy against multi-drug resistant pathogens.

No description available.

No description available.

Acute Lung Injury, characterised clinically as the Acute Respiratory Distress Syndrome is a catastrophic response to a range of pulmonary and non-pulmonary insults. Despite much work the key mechanisms involved in generating the exaggerated immune response that results in lung injury are not completely understood. Hypoxia-inducible factor-1 has been shown to be a key transcription factor in the myeloid cell response to inflammatory signals. The aims of this thesis were to develop a model of acute lung injury and to study the role of Hif-1 in the generation of lung injury in this model. A model of direct pulmonary injury as a result of intratracheal instillation of endotoxin is described. Using this model the role of myeloid cell Hif-1α was characterised using a myeloid cell specific conditional knockout system. The injury in Hif-1α deficient mice was quantitatively similar to the injury seen in wild type animals over a range of time points. However, the quality of the injury, assessed by a measure of nitric oxide mediated damage was reduced. The in vivo data were supported by in vitro studies using a murine macrophage cell line which showed that manipulation of the cellular oxygen tension in the presence of endotoxin alters the ability of the cell to generate nitric oxide. Furthermore, pharmacological manipulation of cellular Hif-1 levels by Dimethyloxallyl Glycine (DMOG) in the macrophage cell increased the generation of nitric oxide in response to endotoxin by altering the expression of a number of the isoforms of Nitric Oxide Synthase. In a final set of experiments the response to intratracheal endotoxin was modulated in mice by the concurrent administration of DMOG. As expected the qualitative response to endotoxin was similar but the NO mediated damage was enhanced in the animals administered DMOG. Manipulation of Hif-1 may have a role in the therapy of lung injury by altering the characteristics of the response.

Introduction: NKT cells are categorised as borderline between NK and T cells, sharing phenotypic and functional characteristics of both cells, demonstrating their capacity to contritube to both pro- or anti-inflammatory processes. However, the role of these cells among lung transplant recipients remains largely unknown. The aim of this study was to determine the role of NKT cells following lung transplantation. Methods: NKT cells were quantified and characterised according to markers of: activation (CD107a, CD161, NKG2D) and immunomodulation (CD200 and CD200R) in peripheral blood and BALs. NKT cell numbers and phenotypes were correlated to clinical variables: immunosuppression, acute rejection, acute infections (viral, bacterial and fungal), bronchiolitis obliterans syndrome (BOS grade), lung function, and demographic variables. Interactions between NKT cells and the transplanted lung were linked by determining the relative expression of immunomodulatory ligand CD200 in lung biopsies. In vitro models were employed to determine the role of NKT cells to acute lung injury, either alone or in combination with cells of the mononuclear phagocyte system (MPS). Results: Higher numbers of immunomodulatory NKT cells (CD200+ and CD200R+) were found as lung function decreased. Data from peripheral blood indicates that recipients whose donors or themselves had been exposed to CMV infection demonstrated increased numbers of NKT cells. Patients with active EBV infections demonstrated higher NKT cell numbers expressing CD200 and CD200R. Data from BALs, indicates that patients with active fungal infections present higher immunomodulatory (CD200R) NKT cells and lower cytotoxicity marker (CD107a). In peripheral blood, lung recipients demonstrated higher NKT cell numbers compared to healthy volunteers. However, the lower relative mean expression of functional markers in the lung transplant group suggests that cells are less active. In vitro cultures with immunosuppressants demonstrated that cell cycle inhibitors (MMF and AZA) and corticosteroids (Prednisolone) are likely to inhibit NKT cell proliferation, while calcineurin inhibitors (Cyclosporine A and Tacrolimus) decrease the relative mean expression of activation markers. Clinical observations indicate that higher doses of Azathioprine may correlate with increased NKT cell numbers and the relative expression of CD200 and CD200R. However, under these conditions the relative expression of activation marker NKG2D decreases. In vitro data from the acute injury model indicates that NKT cells are capable to migrate into the injured lung and become activated following transmigration which is facilitated by the presence of monocytes. We also observed the interaction of NKT cells with endothelial cells, monocytes and macrophages. Also, the relative mean expression of CD200 and CD200R increased at the capillary layer, regardless of injury while upregulation of activation markers (CD107a, CD161 and NKG2D) was found at the capillary layer, following injury. In contrast, the alveolar layer demonstrated a decrease in both activation and immunomodulatory markers, following acute injury. Conclusions: Despite immunosuppression, NKT cells remain present in peripheral blood and BAL following lung transplantation. NKT cell proliferation is likely to be reduced by effect of cell cycle inhibitors, while calcineurin inhibitors exert an immunomodulatory effect. Our data indicates that NKT cells can participate in inflammatory and immunomodulatory events at the alveolar bilayer. Their capacity to infiltrate the lungs was assisted by cells of the mononuclear phagocyte system (MPS), which play an important role in antigen presentation and modulation of acute injury. Further research is needed to elucidate the signals and mechanisms occurring between NKT and MPS interactions and the outcomes these populations drive in acute lung injury.

Mechanically ventilated patients are at risk of developing Ventilator-Associated Lung Injury (VALI). Improved ventilation strategies by lung-protective settings may cause hypercapnia. This study investigated whether attenuation of VALI is attributed to protective ventilation with low tidal volume (VT) or hypercapnia. Lung injury was induced in rats by instillation of 1.25M HCl. Ten rats each were ventilated for 4 hours with: Conventional Normocapnia (highVT), Lung-Protective Ventilation (VT¬ 8mL/Kg), Injurious Normocapnia (highVT, added dead space), Conventional Hypercapnia (highVT, inhaled CO2), Protective Hypercapnia (VT 8mL/Kg, inhaled CO2) and Permissive Hypercapnia (VT 8mL/Kg, hypoventilation). Lung-Protective Ventilation reduced pulmonary edema compared to Conventional and Injurious Normocapnia. Therapeutic hypercapnia reduced alveolar damage and inflammation by reducing IL-6 and MCP-1 in the lung, and IL-1? and TNF-? systemically. Therapeutic hypercapnia may be more effective in attenuating some of the biomarkers of VALI and protecting the lung than protective ventilation alone.

No description available.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A pancreatite aguda (PA) Ã considerada uma situaÃÃo de emergÃncia abdominal, na forma grave da doenÃa os pacientes desenvolvem acentuada resposta inflamatÃria sistÃmica e SÃndrome de DisfunÃÃo de MÃltiplos ÃrgÃos (SDMO). Um terÃo das mortes relacionadas com PA acontecem antes da admissÃo hospitalar, e a maior parte dos casos estÃo relacionados com lesÃo pulmonar aguda (LPA) e sÃndrome do desconforto respiratÃrio agudo (SDRA). Objetivos: Avaliar as alteraÃÃes inflamatÃrias e funcionais do pulmÃo no curso da pancreatite aguda experimental induzida por ceruleÃna. MÃtodos: PA foi induzida em Ratos Wistar, machos pensando 100-150g, pela administraÃÃo de 4 doses de ceruleÃna (20Âg/kg) com intervalo de uma hora e os grupos controle receberam apenas soluÃÃo salina. ApÃs 24 horas, os animais foram sedados, analgesiados e traqueostomizados e anÃlise da funÃÃo pulmonar foi realizada atravÃs da espirometria, onde foram avaliados Fluxo, Volume Corrente (VC), FrequÃncia RespiratÃria (FR) e Volume Minuto (VM), e da mecÃnica pulmonar onde foram observados ElastÃncia DinÃmica (Edin), ComplacÃncia DinÃmica (Cdin), PressÃo de Pico, ResistÃncia (Res). Lavado bronco-alveolar (LBA) foi realizado para contagem total e diferencial de cÃlulas. Amostra de sangue arterial foi colhida para avaliaÃÃo dos parÃmetros gasomÃtricos. Em seguida os animais foram sacrificados e nÃveis sÃricos de amilase, lipase, EPO, TNF-α, GRO-KC, MIP-1, VEGF, IL-1β, IL-2, IL-6, IL-10, IL-12, IL-17, IL-18 e de malondialdeÃdo (MDA) foram medidos. Atividade de mieloperoxidase (MPO) e avaliaÃÃo histolÃgica de pÃncreas e pulmÃo foram determinadas. AlÃm disso, amostras de sangue venoso foram colhidas para avaliaÃÃo de translocaÃÃo bacteriana. Resultados: NÃveis sÃricos de amilase, lipase, citocinas, MDA e atividade de MPO pancreÃtica e pulmonar estavam aumentados nos animais com PA; houve danos ao tecido pancreÃtico e pulmonar, revelados na histologia, nos animais que receberam ceruleÃna, quando comparados ao grupo controle. O LBA dos animais tratados com ceruleÃna demonstrou maior quantidade de cÃlulas, sendo predominantemente macrÃfagos. Gasometria arterial nÃo apresentou diferenÃa significativa entre os grupos. Fluxo, VC e VM se mostraram diminuÃdos nos animais com PA; FR permaneceu inalterada. Edin e PressÃo de Pico estavam maiores e Cdin estava menor nos animais com PA e nÃo houve alteraÃÃes na Res. Estudo da bacteremia foi negativo em ambos grupos. ConclusÃo: CeruleÃna induz PA em ratos com elevaÃÃo dos nÃveis de amilase e lipase pancreÃtica, com alteraÃÃes histopatolÃgicas no pÃncreas e no pulmÃo dependente do infiltrado neutrofÃlico, radicais livres e citocinas inflamatÃrias. PA induz alteraÃÃes espiromÃtricas e na mecÃnica pulmonar que nÃo sÃo dependentes de processo infeccioso. / Acute pancreatitis (AP) is considered an emergency abdominal, the severe form of the disease patients develop intense systemic inflammatory response and Multiple Organ Dysfunction Syndrome (MODS). About one-third of all deaths from acute pancreatitis has been reported to occur prior to admission to hospital, and in most cases, is associated with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Objectives: To assess the inflammatory and functional lung alterations in the course of AP induced by cerulein. Methods: Male Wistar rats (100-150g) were treated four times with one hour interval, intraperitoneally with cerulein (20 μg / kg, suspended in saline) or saline. Twenty-four hours after the first injection of cerulein, the animals were anesthetized, tracheostomized and placed in a spirometer for small animals and with following parameters evaluated: Flow, Volume(VC), Respiratory Frequency(RF) and Minute Volume(MV), and lung mechanics were observed where Dynamic Elastance (Edyn), Dynamic Compliance (Cdyn), Peak Pressure, Resistance (Raw). Bronchoalveolar lavage (BAL) was performed to count and differential cell. Arterial blood sample was drawn for assessment of pulmonary gas exchange parameters. Then the animals were sacrificed and serum amylase, lipase, EPO, TNF-α, GRO-KC, MIP-1, VEGF, IL-1β, IL-2, IL-6, IL-10, IL-12, IL-17, IL-18 and malondialdehyde (MDA) were measured. Myeloperoxidase activity (MPO) and histological evaluation of pancreas and lung were determined. In addition, venous blood samples were collected for evaluation of bacterial translocation. Results: Serum levels of amylase, lipase, cytokines, MDA and MPO activity of pancreatic and lung were increased in animals with PA, there was damage to pancreatic tissue and lung histology revealed, in animals that received cerulein compared to the control group. There was an increase in the number of BAL cells, predominantly macrophages. Arterial blood gas analysis showed no significant difference between groups. Flow, and MV proved lower in animals with PA; FR remained unchanged. Edyn and pressure peak were larger and Cdyn was lower in animals with PA and no changes in Res. There was no translocation in any groups. Conclusion: Cerulein induced AP in rats with elevated serum amylase and pancreatic lipase, with histopathological changes in the pancreas and lung dependent neutrophilic infiltrate, free radicals and inflammatory cytokines. PA induces spirometric and lung mechanics alterations that are not dependent on bacterial translocation

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23071号 / 医博第4698号 / 新制||医||1049(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊達 洋至, 教授 YOUSSEFIAN Shohab, 教授 平井 豊博 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM