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A recently developed therapy for treatment of acute respiratory failure requires that the patient's metabolic carbon dioxide production be eliminated by a membrane oxygenator operated in an extracorporeal blood circuit. In conjunction with peripheral cannulation, the oxygenator should be optimised for CO₂ removal at low blood flow rates of 1.5 ℓ/min or less for adults. An extensive literature survey revealed that very few publications dealt with oxygenator CO₂ performance at low flow rates. Two commercial devices, the Terumo CAPIOX II (1.6 m² and 3.3 m² membrane areas) hollow fibre oxygenator and the Travenol TMO (2.25 m² membrane area) parallel-plate oxygenator were evaluated in relation to the new therapy. A theoretical model describing carbon dioxide transfer in membrane oxygenators was used to correlate the experimental data. The Terumo CAPIOX II 3.3 m² unit was the only device capable of satisfying the carbon dioxide removal requirements necessary for the new therapy at the low blood flow rates stipulated. Effects of blood and gas flow maldistribution were also studied in the TMO and CAPIOX II units respectively. Non-uniform blood flow was not a major factor contributing to the decline in CO₂ transfer performance compared with theory. This was confirmed in experiments with a modified TMO unit. Comparison with theory indicated that the membrane resistance was the controlling factor for CO₂ transfer in the CAPIOX II device. A method was developed to assess the CO₂ transmission rate (Gco₂) through oxygenator membranes under gas-membrane-liquid contact conditions. This forms the basis for the selection of suitable membrane materials for oxygenators. Although the GCO₂ values for homogeneous silicone rubber membranes were consistent with the results of previous workers, significantly higher values were obtained for microporous polypropylene membranes. For microporous membranes under liquid contact conditions a 5-fold reduction in GCO₂ is obtained in this study compared to gas-membrane-gas tests, indicating that micropore wetting imposes a significant resistance to CO₂ transfer.

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.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in partial fulfilment of the requirements for the degree of Master of Medicine in the branch of Internal Medicine. Johannesburg, 2014 / BACKGROUND: Interstitial lung disease (ILD) is one of the leading causes of death in systemic sclerosis (SSc). PATIENTS AND METHODS: A retrospective review of case records, over 20 years, of SSc patients attending a tertiary Connective Tissue Diseases Clinic. Comparisons between ILD and non-ILD groups at presentation were performed in order to identify baseline associations and predictors of ILD. RESULTS: Of the 151 participants that met inclusion criteria, 60 (40%) had ILD. On multivariate analysis the only three variables to remain significant were median duration of disease (OR 1.2 (1.1-1.3); p<0.001), speckled anti-nuclear antibody (ANA) pattern (OR 2.95 (1.22-7.15); p=0.017) and bibasal crackles (OR 5.4 (2.1- 13.5); p<0.0001). Univariate analysis of baseline variables associated with interstitial lung disease in systemic sclerosis. Baseline Variable ILD (n=60) Non-ILD (n=91) OR (CI 95%) p Bibasal crackles (%) 28 (46.7) 10 (11.0) 7.1 (3.1-16.3) <0.0001 Diffuse disease subtype (%) 49 (81.7) 45 (48.9) 4.6 (2.1-9.9) <0.001 Limited disease subtype (%) 8 (13.3) 38 (41.3) 0.2 (0.1-0.5) <0.001 Anti-centromere antibodies (%) 0 (0.0) 10 (13.0) - 0.006 Cough (%) 21 (35.0) 15 (16.5) 2.7 (1.3-5.9) 0.007 Median duration in years (IQR) 6.1 (8.3) 4.0 (5.0) 2.2 (1.8-2.4) 0.009 Speckled ANA pattern (%) 29 (50.9) 25 (32.5) 2.5(1.2-4.9) 0.010 Dyspnoea (%) 27 (45.0) 24 (26.4) 2.3 (1.1-4.6) 0.014 Gold mining history (%) 5 (8.3) 1 (1.1) 8.2 (0.9-71.9) 0.037 ANA=antinuclear antibody; ILD=interstitial lung disease; IQR= interquartile range; OR=odds ratio Additionally, dyspnoea was associated with ILD severity (p=0.008). Bibasal crackles (p=0.014), increased plasma urea (p=0.041), and reduced serum albumin (p=0.007) were associated with mortality in the ILD group. CONCLUSION: Interstitial lung disease in South African SSc patients is common. The diffuse cutaneous disease subtype appears to drive the disease process. There should be a high index of suspicion for ILD in SSc patients presenting with a gold mining history, dyspnoea, cough and bibasal crackles.

Lung diseases are one of the leading causes of mortality and morbidity worldwide. Understanding these diseases at a molecular level remains a critical component to developing effective therapeutics. Previous work has shown that gene expression alterations play an important role in disease initiation, maintenance, and progression as well as serve as diagnostic tools in disease. However, much remains to be uncovered regarding the role that microRNAs play in both healthy and diseased lung tissue. This thesis seeks to utilize methods of bioinformatics, cell biology, and molecular biology to examine the effect of miR-4423 on lung epithelial cell differentiation (Aim 1), miR-424 on never smoker derived lung adenocarcinoma (Aim 2), and miR-34c isomiRs in interstitial lung disease (ILD) (Aim 3). First, we examined the role of miR-4423 in lung mucociliary epithelium by employing the use of an air-liquid interface culture system, finding miR-4423 has an effect in ciliated cell differentiation and that a loss of miR-4423 is associated with cancer progression. These findings suggest that miR-4423’s actions in airway epithelium differentiation may potentially provide a therapeutic role in lung cancer. Next, we validated transcriptomic differences between lung tumor tissues resected from never and ever smokers. Specifically, miR-424, a predicted regulator of a large number of gene expression changes in never smoker lung adenocarcinoma, was found to regulate cell migration, potentially identifying a novel target and/or pathway for therapeutic action. Lastly, the function of microRNA isomiRs is relatively unknown. We validated miR-34c as upregulated in ILD and modulated both miR-34c and a miR-34c 5’ isomiR in lung relevant cell lines to explore their differing biological roles. We found that they are capable of targeting differing mRNA, indicating an independent role for isomiRs in disease. The studies contained in this dissertation offer valuable insight into the biology of microRNAs in the lung and how they might be employed as therapeutic targets for a number of common lung diseases. In addition, biological insights into the complexity of microRNAs in the lung highlight the need to better understand diseases influenced by microRNA expression and microRNA variants in regards to actionable therapeutics. / 2017-12-01T00:00:00Z

Neutrophils are a central component of the innate immune system, whose major role is to defend the host against invading microorganisms. As such they are integral players in the process of inflammation, the response of vascular tissues to injury. They are frequently the first immune cells recruited from the systemic circulation into a site of tissue injury or infection where they themselves play a key antimicrobial role. Direct killing of microbes can be accomplished by phagocytosis, degranulation, production of reactive oxygen species (ROS) or the release of DNA and antimicrobial peptides into the extracellular milieu (NETosis). In addition neutrophils orchestrate the recruitment and activation of other leucocytes, further contributing to host defence. The central importance of neutrophils in immunity is revealed by defects in either number or function leading to recurrent life threatening infection. However, as the toxic arsenal of neutrophil constituents lack specificity they can also be damaging to surrounding host tissues causing exacerbated inflammation. It is therefore essential that neutrophil function is tightly controlled to allow an appropriate response to be mounted against invading pathogens while simultaneously minimising host tissue injury. Therefore, once the inciting inflammatory insult has been successfully cleared or controlled it is imperative that these non-tissue resident specialised immune cells are rapidly ‘switched off’ or cleared to allow the return to homeostasis. This resolution phase of the inflammatory cascade is now recognised as an energy dependent, finely controlled endogenous process, the beginnings of which are activated at the onset of inflammation. One of the main aims of resolution is to ensure efficient clearance of leucocytes that are no longer necessary. It is likely that a major clearance route is by the highly regulated and energy dependent processes of neutrophil programmed cell death (apoptosis) with subsequent uptake and disposal of apoptotic neutrophils by tissue macrophages. This process of neutrophil apoptosis renders the neutrophils nonfunctional and preserves cell membrane integrity, thus preventing further release of histotoxic neutrophil-derived inflammatory mediators into the extracellular environment. Furthermore, the recognition, uptake and disposal of apoptotic neutrophils cause a dynamic change in the phagocytosing macrophage phenotype with alterations in inflammatory mediator production. The fundamental importance of neutrophil apoptosis and subsequent efferocytosis in inflammation resolution is highlighted by the pathological consequences of neutrophil necrosis or failed apoptotic cell clearance, which leads to enhanced tissue injury and autoimmunity. Acute lung infection (pneumonia) is a common and serious condition affecting both developed and developing countries; globally, childhood pneumonia is the leading cause of death in children aged less than 5 years and pneumonia is the most common fatal infection in the developed world. In over half of patients with community acquired pneumonia no causative organism is ever isolated suggesting that although the immune response has successfully controlled infection, continued uncontrolled neutrophilic inflammation in the lung continues to cause morbidity and mortality. Indeed, pneumonia frequently progresses to acute respiratory distress syndrome (ARDS), a devastating acute inflammatory condition of the lungs characterized by inflammatory cell recruitment and accumulation of protein rich oedema fluid leading to impaired lung function. ARDS affects 200,000 critically ill patients in the USA per year, and has a substantial mortality rate of up to 40%. Despite advances in intensive care treatment and antimicrobial therapy mortality from pneumonia has not fallen since the 1950s, and at present there are no specific therapies for infection-related lung inflammation or ARDS. Understanding the mechanism behind such uncontrolled, persisting inflammation, and the need for novel approaches to target infection related lung injury are therefore both urgent and essential. This thesis examines the potential of neutrophil apoptosis-inducing pharmacological agents as potential treatments for infection-associated lung inflammation. The primary agents used include a cyclin-dependent kinase inhibitor as well as plant-derived polyphenolic flavones. The ability of these compounds to induce human neutrophil apoptosis in vitro, the key importance of the intracellular neutrophil survival protein Mcl-1 in mediating this process, and the effect of targeting Mcl-1 in human macrophages is investigated. In addition, neutrophilic inflammation is modelled in zebrafish and mice with both sterile and bacterial-driven models of inflammation. A key role for Mcl-1 is delineated in vivo, with it acting as an endogenous controller of the innate immune response by influencing neutrophil apoptosis, but without effects on macrophage apoptosis or ability to phagocytose apoptotic cells. Driving neutrophil apoptosis by down-regulation of Mcl-1 accelerates resolution of inflammation in vivo. This therapeutic approach is also found to have indirect anti-bacterial effects in a model of E. Coli induced pneumonia, in stark contrast to established anti-inflammatory approaches which routinely cause immune paresis and life threatening infection. As such, targeting inflammatory cell apoptosis by changes in Mcl-1 offers a potential new therapeutic approach for the treatment of infection-associated inflammation.

The work in this thesis describes the approaches taken to advance the field of pulmonary optical molecular imaging for the diagnosis of unexplained pulmonary opacities in the critically ill patient where bacterial causes are suspected and the investigation of pulmonary nodules and masses where lung cancer is suspected. The bacterial work includes the development and assessment of a multivalent fluorescently labelled antimicrobial peptide fragment that allows for the in vivo in situ detection of bacteria in the distal lung. This Smartprobe (chapter 3), called NBD-UBIdend remains specific for bacteria and pathogenic pulmonary fungi (Aspergillus fumigatus) over mammalian cells, and has a clinically relevant limit of detection when it is imaged in an ex vivo whole lung ovine ventilated model using fibered confocal fluorescence microscopy (FCFM). Furthermore, NBD-UBIdend detects all bacteria assessed, including a panel that accounts for 70% of ventilator associated pneumonia causing organisms. Chapter 4, develops this further and describes the in vitro and ex vivo evaluation of another Smartprobe utilising a fluorescently labelled modified polymyxin B moiety, called NBD-PMX. This compound detects gram-negative but not gram-positive bacteria and is compatible with pulmonary FCFM. This combination of Smartprobes and FCFM could allow the immediate stratification of patient therapy in the assessment of pulmonary opacities where bacterial causes are suspected. The lung cancer work includes the use of label free FCFM in a clinical cohort to determine if autofluorescence patterns can differentiate benign and malignant pulmonary nodules. This work (chapter 5) demonstrates here there is no differentiation using FCFM alone and therefore, for this technology to be used in lung cancer diagnostics a Smartprobe strategy may be beneficial. Finally, chapter 6 demonstrates a Smartprobe based approach for interrogating lung cancer and discusses a matrix metalloproteinase (MMP) compound that detects MMPs in a whole ventilated lung utilising a modified spontaneous ovine pulmonary adenocarcinoma model.

acase@tulane.edu / Influenza is a highly contagious viral respiratory infection that occurs in annual outbreaks. Activity levels for the 2017-2018 influenza season reached heights not seen since the 2009 pandemic. This was partly due to the inefficiency of the vaccine (25% effective) against the predominant circulating strain, H3N2. To make matters worse, current antiviral therapies must be given within 48 hours of the onset of symptoms. This is often well outside the window of opportunity for hospitalized patients. Developing a therapy that promotes repair of the extensive damage that occurs in severely infected patients is vital for their recovery. Our lab focuses on the innate immune response, more specifically the IL-22 pathway, and the mechanisms involved in repair following pulmonary injury and infection. IL-22 is important in cell proliferation, wound healing, maintaining epithelial barriers and innate pathogen defense. In the lung, its receptor, IL-22Ra1, is only found on epithelial cells and is rapidly induced in response to damage of the lung epithelium. The central hypothesis of this dissertation is that IL-22Ra1 is induced during influenza infection on pulmonary epithelial and progenitor cells, allowing for enhanced sensitivity to IL-22. We have found this induction to be TLR3 and STAT1 dependent. In vivo, bronchial brushings from H1N1 infected mice (PR/8/34) mice demonstrate that Il-22ra1 is rapidly induced in the airways following infection. This occurs in a STAT1 dependent manner as upregulation does not occur in STAT1-/- mice in vivo or following STAT1 inhibition in vitro. This pathway is important as IL-22 treatment induces expression of tight junction transcripts both in vitro and in vivo. Moreover, we believe this induction of IL-22Ra1 is critical for the survival of lung progenitor cells as we have data showing that over 80% of basal cells express Il-22ra1 in the naïve lung. Furthermore, we have developed a lung organoid model and upon treatment with IL-22, organoid size was significantly increased after seven days as evidenced by measurement and BrdU incorporation. Overall, our data shows that IL-22Ra1 is highly induced after injury and subsequent treatment with IL-22 is essential for altering tight junctions and promoting lung repair. / 1 / Kelly Douglas Hebert II

The presence of cholesterol is critical in defining a dynamic lateral structure in pulmonary surfactant membranes, including the segregation of fluid-ordered and fluid-disordered phases. However, an excess of cholesterol has been associated with impaired surface activity both in surfactant models and in surfactant from injured lungs. It has also been reported that surfactant protein SP-C interacts with cholesterol in lipid/protein interfacial films. In the present study, we have analyzed the effect of SP-C on the thermodynamic properties of phospholipid membranes containing cholesterol and on the ability of lipid/protein complexes containing surfactant proteins and cholesterol to form and re-spread interfacial films capable of producing very low surface tensions upon repetitive compression-expansion cycling. We have also analyzed the effect of cholesterol on the structure, orientation and dynamic properties of SP-C embedded in physiologically relevant model membranes.