Zugl.: Giessen, Univ., Diss., 2005

Universiẗat, Diss., 2005--Giessen.

Zugl.: Giessen, Universiẗat, Diss., 2008.

Zugl.: Giessen, Universiẗat, Diss., 2008.

Marburg, Univ., Diss., 2009

Neutrophils as key effector cells of the innate immune system migrate from the circulation into sites of inflammation and are essential for the containment, killing and clearance of invading pathogens through a variety of highly regulated cell functions. Despite this beneficial role their involvement can also be detrimental. In a number of diseases dysregulated neutrophil influx and activation results in significant tissue damage and worsening of the acute inflammatory event as well as long term tissue injury, scarring and fibrosis. One such pulmonary condition is acute respiratory distress syndrome (ARDS) which, despite decades of intensive research and multiple clinical trials, remains without a cure and has an associated mortality rate of approximately 40%. Delineating and understanding the key pathogenic mediators that drive neutrophil recruitment into the lung in the context of both bacterial and sterile injury is therefore vital in the development of novel therapies. Neutrophils migrate towards a variety of agents but amongst such factors a hierarchy exists with bacterial-derived products, including formylated peptides, dominant in this process. In sterile tissue injury where no bacterial factors are present the mediators involved change but a hierarchy still exists. Mitochondrial formylated peptides are released following necrotic cell death and bind to formyl peptide receptor 1 (FPR1) on the neutrophil surface inducing migration and activation. Like bacterial formylated peptides they are powerful chemoattractants and are therefore likely to be important in recruiting neutrophils to sites of injury and inflammation. Hypothesis: The central hypothesis of this thesis is that mitochondrial formylated peptides, as end-target chemoattractants, are elevated in patients with ARDS and drive neutrophil recruitment through binding to FPR1. Inhibition of FPR1 in models of acute lung injury will therefore result in attenuation of this inflammatory response through multiple FPR1-mediated effects implicating both formylated peptides and their cognate receptor in the pathogenesis of sterile ARDS. Results: Free mitochondrial DNA and formylated peptides were elevated in the circulation of patients with ARDS or severe paracetamol-induced hepatic failure relative to healthy controls. In addition, FPR1 receptor number was increased on the surface of neutrophils isolated from critically ill septic patients. Isolated mitochondrial formylated peptides induced FPR1- dependent chemotaxis in primary human neutrophils in vitro. Alongside this, FPR1 ligand binding resulted in increased cell surface β2-integrin expression [integrin alpha M beta 2 (ITGB2); also called CD11b/CD18, Mac-1 or CR3] through intracellular activation of PI-3Kand MAPK-dependent signalling pathways. Indeed, blockade of neutrophil cell-surface integrin alpha M (ITGAM; also known as CD11b)) resulted in a reduction in mitochondrial formylated peptide-induced chemotaxis. To determine the production of human neutrophil IL-1β, a pivotal chemokine within a sterile inflammatory environment, a novel method for the in vitro isolation of ultrapure neutrophils was developed. Neutrophils were isolated by autofluorescence-based flow sorting as determined by intrinsic differences in neutrophil and eosinophil autofluorescence and their size and granularity relative to circulating mononuclear cells. Analysis of this approach demonstrated the ability to rapidly collect a highly pure neutrophil population (99.95 ± 0.03%). Flow sorting did not alter the activation state or functional capacity of these cells relative to unsorted neutrophils with regards to several measures of neutrophil behaviour/function. Cells also remained fully responsive to a variety of neutrophil agonists with no evidence of neutrophil priming. The capacity of highly pure neutrophils to secrete IL-1β was determined to be approximately 160-fold lower than equivalent numbers of circulating peripheral blood mononuclear cells. In the context of an inflammatory environment however this is likely to be of biological significance given the large number of infiltrating neutrophils. In sterile hydrochloric acid-induced acute lung injury pharmacological inhibition of FPR1 with cyclosporin H (CsH), or use of transgenic FPR1-/- mice, resulted in inhibition of neutrophil migration into the alveolar space 24 hours after injury. This was associated with a reduction in pulmonary haemorrhage, extravascular protein leak and pro-inflammatory cytokine expression with improved histological appearances. Furthermore, the HCl acid-induced reduction in alveolar macrophage numbers was inhibited by CsH with interstitial macrophages displaying an alternatively activated phenotype. Importantly, delivery of CsH 12 hours after the onset of injury also reduced acute lung inflammation demonstrating its potential therapeutic relevance in the treatment of human disease. In non-sterile E. coli-mediated acute lung injury partial antagonism of FPR1 with CsH resulted in a reduction in neutrophil migration and vascular leak with no effect on pulmonary bacterial load. A narrow therapeutic window existed however as increased concentrations of CsH, or infection in FPR1-/- mice, resulted in a reduction in alveolar neutrophil number and increase in E. coli at 24 hours. Alongside effects on myeloid cells within the lung FPR1 was found to be expressed on mouse lung epithelial cells. A technique to isolate and culture mouse type 1 alveolar epithelial (AT1) cells was therefore developed. Flow sorting of anti-type 1 alpha (anti-T1α) stained single cell lung homogenates with subsequent culture on transwell membranes resulted in the development of confluent AT1 cell monolayers after 10 days. Formylated peptides appear to induce a reduction in transepithelial resistance and increase in permeability across a monolayer in vitro alongside an increase in release of the neutrophil chemo-attractant mouse CXCL8 (KC). Conclusions: Taken together, mitochondrial formylated peptides released following cell necrosis and FPR1 play a significant role in the pathogenesis of sterile acute lung injury. This is likely to be predominantly through neutrophil-dependent means but data presented here also suggests that their role in macrophage function and alveolar epithelial cell permeability may be important. Inhibition of FPR1 may therefore represent a novel and multi-cellular therapeutic target in the treatment of ARDS.

Background: With the COVID-19 pandemic, there has been an influx of patients with acute respiratory distress syndrome (ARDS), an inflammatory lung condition. ARDS survivors are at high risk for developing post-traumatic stress disorder (PTSD) due to intensive care unit (ICU) medical treatments/procedures. They are known to have traumatic memories triggered by their sensorium months to years after being discharged from the ICU. One study found that 23% of ARDS survivors experienced long-term PTSD symptoms 2-3 years after hospital discharge (Bienvenu et al., 2018). Unknown is whether there are similarities in the memories and sensory triggers of PTSD amongst ARDS and COVID ARDS survivors. Purpose: The purpose of this study was to 1) identify the most common vivid ICU memories and sensory triggers for PTSD symptoms in survivors of ARDS and COVID positive ARDS; 2) to analyze the frequency of sensory triggers and determine whether differences exist between ARDS and COVID ARDS survivors. Method: A multi-step, thematic analysis of qualitative data from 27 patients was completed (20 COVID ARDS patients and 7 ARDS patients) by a team of 7 researchers. Patients were asked a series of open-ended questions regarding vivid memories and sensory triggers for them. Major themes were generated from their responses. Results: Major themes identified were prevalent in both COVID ARDS and ARDS groups. Prominent vivid memories included medical treatment/procedures, emergence delirium, illusions/hallucinations, vivid nonsense dreams and sensory to dream conversion. Common sensory triggers included seeing medical equipment, hearing beeping/alarms, seeing media depictions of the hospital setting, hospital smells and seeing doctors, nurses, hospitals. Differences between COVID-ARDS and ARDS groups were not notable. Conclusion: The data collected in this study revealed ARDS and COVID ARDS patients experience sensory inputs during their ICU stay that contribute to the development of vivid, long-lasting memories and subsequent PTSD symptoms. Survivors' everyday lives are altered by these symptoms, impacting their ability to work, familial relationships, and likelihood to seek out healthcare. Data from this study is being used in a compressed exposure therapy trial and should be incorporated into future PTSD preventative and treatment interventions.

No description available.

The cardiopulmonary effects of endotoxin administration (1 microgram/kg) were evaluated in 8-10 week old SPF-derived Yorkshire pigs, both because endotoxemia is a common and important swine problem, and because the pig is a good model for human adult respiratory distress syndrome. Physiological changes included sustained increases in mean pulmonary artery pressure, pulmonary vascular resistance, pulmonary arterial wedge pressure, heart rate, hematocrit, and the arterial partial pressure of carbon dioxide. Transient increases were also observed in central venous pressure and airway pressure. Transient increases, followed by decreases, were observed in mean systemic arterial pressures and systemic vascular resistance. Decreases were seen in cardiac output, cardiac index, arterial partial pressure of oxygen and oxygen saturation. The number of circulating leukocytes, lymphocytes and segmented neutrophils decreased with endotoxin infusion. To investigate the role of airway smooth muscle, bronchial rings were isolated and exposed to contractile agents in tissue baths. A hyperresponsiveness of the third generation bronchi to substance P, carbachol, bradykinin and electric field stimulation was observed. However the increase in response to bradykinin and electric field stimulation were not statistically significant. Histopathology of the lungs demonstrated congestion, hemorrhage and neutrophilic infiltration. / Master of Science

In der vorliegenden Arbeit wird beschrieben, wie intrapulmonal stufenweise per Instillation gegebenes, hochgereinigtes Perfluorocarbon (FC 3280) die Hämodynamik, die Lungenfunktion und die Überlebenszeit bei ARDS im Tierexperiment beeinflusst. An zwölf anästhesierten wurde eine prospektive, randomisierte und kontrollierte Studie durchgeführt. Bei den Tieren wurde durch wiederholte Lavagen mit warmem Kochsalz ein Surfactantmangel erzeugt. Anschließend wurden sechs Tiere mit partieller Flüssigkeitsbeatmung (PLV) durch zweimalige Applikation von 7,5 ml/kgKG FC 3280 behandelt (Behandlungsgruppe). In der Kontrollgruppe erhielten 6 Tiere keine weitere Therapie. Die Hämodynamik und der Gasaustausch wurden alle 30 Minuten nach Gabe des PFC kontrolliert. In der Behandlungsgruppe lag der PaO2 nach 7,5 ml/kgKG PFC bei 67 +- 33 mmHg und nach insgesamt 15 ml/kgKG bei 106 +- 74 mmHg. Drei der Tiere sprachen dabei gut auf die Therapie an. Bei ihnen konnten beachtliche Verbesserungen in der Sauerstoffversorgung gezeigt werden. So verbesserte sich der PaO2 von 62 mmHg auf 450 mmHg bei einem der Tiere, welches gut auf PLV ansprach. In der Kontrollgruppe blieb der Gasaustausch jedoch unverändert. Die hämodynamischen Parameter verhielten sich während der ersten 6 Stunden stabil in der Behandlungsgruppe während sich bei den Kontrolltieren die Verhältnisse zunehmend verschlechterten. In der Behandlungsgruppe lag das Mittel der Überlebenszeit bei 8,2 +- 4,5 Std. und bei 1,8 +- 1,4 Std. in der Kontrollgruppe (p < 0,05). Zusammenfassend lässt sich sagen, dass die partielle Flüssigkeitsbeatmung mit Perfluorocarbon nicht bei allen Tieren im experimentellem Lungenversagen durch Lavage die arteriellen Oxygenierung bessert. Jedoch auch die Tiere die keinen Anstieg des PaO2 zeigten, lebten länger als die Kontrolltiere. Somit zeigt die verbesserte Überlebenszeit durch PLV einen positiven Effekt in der Behandlung des akuten Lungenversagens. / This thesis discusses the effects of intrapulmonal application of a highly purified perfluorocarbon (FC3280) concerning haemodynamics, gas exchange and survival rates in an experimental animal model of ARDS. A randomised, controlled and prospective study was performed on twelve anesthetised pigs. Repetitive saline lunglavage resulted in surfactant depletion. Subsequently six pigs received partial liquid ventilation with two aliquots of 7,5 ml/kg FC3280 (treatment group), whereas 6 pigs received no further treatment (control group). Haemodynamic values and gas exchange were recorded in a 30 minutes interval. Within the treatment group the PaO2 after 7, 5 ml/kg PFC was 67 +- 33 mmHg and after 15 ml/kg 106 +- 74 mmHg. Three animals responded well to the treatment. The responding animals showed improvements of arterial oxygen contents. One pig showed a increase in PaO2 from 62 mmHg to 450 mmHg. Within the control group gas exchange remained unchanged. Haemodynaimc values in the treatment group remained stable over six hours but deteriorated in the control group. Survival rates were 8,2 +- 4,5 hours in the treatment group and 1,8 +- 1,4 hours in the control group. In conclusion our study showed that partial liquid ventilation improved oxygenation not in all animals treated with perfluorocarbons. While these animals showed no improved in oxygenation they survived longer than the controls. The significant improvement in survival time shows a positive effect in the treatment of ARDS.