Ischemia-reperfusion (IR) injury is a severe complication in lung transplantation characterized by inflammation, alveolar damage, and hypoxemia. Alpha 1-antitrypsin (A1AT), a protease inhibitor, is currently used clinically for the treatment of A1AT deficiency emphysema. A1AT has been shown to have the potential to reduce IR injury through its anti-inflammatory and anti-apoptotic effects. We hypothesized that A1AT will ameliorate IR injury through these effects. We tested A1AT in two models of IR: a cell culture model of simulated lung transplantation and a rat in situ pulmonary ligation model. In cell culture, we found that A1AT exerts its protective effects by inhibiting cell death and inflammatory cytokine release in a dose-dependent manner. In the rat pulmonary ischemia-reperfusion model, we found that A1AT improved lung function by inhibiting apoptosis and inflammation. There is potential for future application of A1AT in the treatment of IR injury in lung transplantation.

Ischemia-reperfusion (IR) injury is a severe complication in lung transplantation characterized by inflammation, alveolar damage, and hypoxemia. Alpha 1-antitrypsin (A1AT), a protease inhibitor, is currently used clinically for the treatment of A1AT deficiency emphysema. A1AT has been shown to have the potential to reduce IR injury through its anti-inflammatory and anti-apoptotic effects. We hypothesized that A1AT will ameliorate IR injury through these effects. We tested A1AT in two models of IR: a cell culture model of simulated lung transplantation and a rat in situ pulmonary ligation model. In cell culture, we found that A1AT exerts its protective effects by inhibiting cell death and inflammatory cytokine release in a dose-dependent manner. In the rat pulmonary ischemia-reperfusion model, we found that A1AT improved lung function by inhibiting apoptosis and inflammation. There is potential for future application of A1AT in the treatment of IR injury in lung transplantation.

Acute lung injury is marked by profound neutrophil influx along with fluid accumulation that impairs lung function at the cost of high mortality (up to 40%). Neutrophils are activated and their constitutive apoptosis is inhibited during this phase in order to be competent phagocytes over the next few hours. Activated neutrophils release copious amounts of toxic mediators that cause tissue damage leading to impaired barrier function and finally, impaired lung function. Therefore, one of the critical needs is to identify molecules that regulate neutrophil migration and silence activated neutrophils to prevent exuberant tissue damage. Angiostatin is an anti-angiogenic molecule highly expressed in lavage fluid of patients with acute respiratory distress syndrome. Angiostatin has recently been shown to inhibit neutrophil infiltration in mice peritonitis. However, the role of angiostatin in modulating neutrophil physiology and lung inflammation remains unknown. I studied the role of angiostatin, an anti-angiogenic molecule, in neutrophil activation and recruitment <i>in vivo</i> and <i>in vitro</i>. Angiostatin was endocytosed only by activated neutrophils, inhibited neutrophil polarity in fMLP-activated neutrophils probably through integrin &alpha;_V&beta;₃, and inhibited MAPK signalling in LPS-activated neutrophils. Angiostatin suppressed formation of reactive oxygen species and activated caspase-3 in neutrophils in both pre-and post-LPS treatments. Finally, angiostatin reduced adhesion and emigration of neutrophils in post-capillary venules of TNF&alpha;-treated cremaster muscle. The next study was designed to investigate the role of angiostatin in acute lung injury. I used <i>E. coli</i> lipopolysaccharide induced acute lung injury mouse model to test the effects of angiostatin through analyses of bronchoalveolar lavage and lung tissues. In addition, I made novel use of synchrotron diffraction enhanced imaging of mouse lungs to assess lung area and contrast ratios over 9 hours as surrogates for lung inflammation. Subcutaneous treatment with angiostatin reduced neutrophil influx, protein accumulation, lung Gr1+ neutrophils and myeloperoxidase activity, phosphorylated p38 MAPK without affecting the levels of MIP-1&alpha;, IL-1&beta;, KC and MCP-1 in lavage and lung homogenates. Diffraction enhanced imaging showed that angiostatin causes a time-dependent improvement in lung area and lung contrast ratios that reflect improvement in lung edema. Overall, the study shows that angiostatin is a novel inhibitor of acute lung injury in mice. Moreover, DEI offers a highly useful technique in evaluating dynamics of lung inflammation and to investigate the therapeutic impact of new drugs on lung inflammation. I conclude that angiostatin is a novel inhibitor of neutrophil migration, activation and acute lung injury.

Immune cells such as neutrophils and monocytes enter tissues after tissue damage and clear cell debris to allow repair cells such as fibroblasts to close the wound. Monocytes also differentiate into fibroblast-like cells called fibrocytes to mediate wound healing, similar to fibroblasts. However, in abnormal wound healing such as acute respiratory distress syndrome (ARDS) and fibrosing diseases, the accumulation of immune cells such as neutrophils or fibrocytes become detrimental to health. In ARDS, neutrophils accumulate in the lungs and causes additional damage by producing reactive oxygen species (ROS). In fibrosing diseases, increased fibrocyte differentiation is one of the causes that increase extracellular matrix deposition, which leads to severe scar tissue build up. Since there are no effective treatments for ARDS or fibrosing diseases, understanding the regulation of neutrophil activation or fibrocyte differentiation could be helpful to develop new effective therapies. The Gomer lab has found several factors that either promote or inhibit fibrocyte differentiation. The pro-fibrotic cytokines such as IL-4 and IL-13 potentiate fibrocyte differentiation while the plasma protein serum amyloid P (SAP), crosslinked IgG, and the pro-inflammatory cytokines IFN-γ and IL-12 inhibit fibrocyte differentiation. In this thesis, I have now shown that additional factors such as toll-like receptor 2 (TLR2) agonists and low molecular weight hyaluronic acid (LMWHA) inhibit fibrocyte differentiation, while high molecular weight hyaluronic acid (HMWHA) potentiate fibrocyte differentiation. The accumulation of neutrophils in the lungs is one of the major factors that debilitate the health of a patient in ARDS. Since neutrophils have Fc receptors, I examined the effect of SAP on neutrophil spreading, adherence, activation, and accumulation. SAP inhibits neutrophil spreading induced by cell debris and TNF-α induced adhesion, but SAP is unable to have any effect on classic neutrophil adhesion molecules or the production of hydrogen peroxide. SAP inhibits neutrophil accumulation in the lungs of bleomycin-injured mice. There is an exciting possibility of using SAP as a therapeutic agent to treat ARDS.

Acute lung injury is marked by profound neutrophil influx along with fluid accumulation that impairs lung function at the cost of high mortality (up to 40%). Neutrophils are activated and their constitutive apoptosis is inhibited during this phase in order to be competent phagocytes over the next few hours. Activated neutrophils release copious amounts of toxic mediators that cause tissue damage leading to impaired barrier function and finally, impaired lung function. Therefore, one of the critical needs is to identify molecules that regulate neutrophil migration and silence activated neutrophils to prevent exuberant tissue damage. Angiostatin is an anti-angiogenic molecule highly expressed in lavage fluid of patients with acute respiratory distress syndrome. Angiostatin has recently been shown to inhibit neutrophil infiltration in mice peritonitis. However, the role of angiostatin in modulating neutrophil physiology and lung inflammation remains unknown. I studied the role of angiostatin, an anti-angiogenic molecule, in neutrophil activation and recruitment <i>in vivo</i> and <i>in vitro</i>. Angiostatin was endocytosed only by activated neutrophils, inhibited neutrophil polarity in fMLP-activated neutrophils probably through integrin &alpha;_V&beta;₃, and inhibited MAPK signalling in LPS-activated neutrophils. Angiostatin suppressed formation of reactive oxygen species and activated caspase-3 in neutrophils in both pre-and post-LPS treatments. Finally, angiostatin reduced adhesion and emigration of neutrophils in post-capillary venules of TNF&alpha;-treated cremaster muscle. The next study was designed to investigate the role of angiostatin in acute lung injury. I used <i>E. coli</i> lipopolysaccharide induced acute lung injury mouse model to test the effects of angiostatin through analyses of bronchoalveolar lavage and lung tissues. In addition, I made novel use of synchrotron diffraction enhanced imaging of mouse lungs to assess lung area and contrast ratios over 9 hours as surrogates for lung inflammation. Subcutaneous treatment with angiostatin reduced neutrophil influx, protein accumulation, lung Gr1+ neutrophils and myeloperoxidase activity, phosphorylated p38 MAPK without affecting the levels of MIP-1&alpha;, IL-1&beta;, KC and MCP-1 in lavage and lung homogenates. Diffraction enhanced imaging showed that angiostatin causes a time-dependent improvement in lung area and lung contrast ratios that reflect improvement in lung edema. Overall, the study shows that angiostatin is a novel inhibitor of acute lung injury in mice. Moreover, DEI offers a highly useful technique in evaluating dynamics of lung inflammation and to investigate the therapeutic impact of new drugs on lung inflammation. I conclude that angiostatin is a novel inhibitor of neutrophil migration, activation and acute lung injury.

Introduction: Currently, there are no specific pharmacological treatments for sepsis-induced acute respiratory distress syndrome (ARDS). And mesenchymal stem cells (MSCs) have shown reparative potential in both sepsis and ARDS. Objectives: To determine the role of MSC administration in the modulation of pulmonary host-responses to sepsis via differential regulation of regulatory microRNAs (miRNAs/miRs). Methods: MicroRNA and mRNA profiling was performed to identify differential expression. Quantitative real time polymerase chain reaction (qRT-PCR), trans-endothelial electrical resistance (TEER) measurements, and luciferase activity assay were used. Results: MicroRNA expression was examined in Human Pulmonary Microvascular Endothelial Cells (HPMECs). One miRNA – miR-193b-5p, targets occludin, a tight junction protein associated with endothelial leakage. A specific regulatory relationship between miR-193b-5p and occludin was identified. The loss in endothelial integrity was rescued when miR-193b-5p inhibitor was transfected. Conclusion: miR-193b-5p is a suppressor of occludin. Studying transcriptional changes allows identification of therapeutically relevant mediators for ARDS/ALI treatment.

Introduction: Currently, there are no specific pharmacological treatments for sepsis-induced acute respiratory distress syndrome (ARDS). And mesenchymal stem cells (MSCs) have shown reparative potential in both sepsis and ARDS. Objectives: To determine the role of MSC administration in the modulation of pulmonary host-responses to sepsis via differential regulation of regulatory microRNAs (miRNAs/miRs). Methods: MicroRNA and mRNA profiling was performed to identify differential expression. Quantitative real time polymerase chain reaction (qRT-PCR), trans-endothelial electrical resistance (TEER) measurements, and luciferase activity assay were used. Results: MicroRNA expression was examined in Human Pulmonary Microvascular Endothelial Cells (HPMECs). One miRNA – miR-193b-5p, targets occludin, a tight junction protein associated with endothelial leakage. A specific regulatory relationship between miR-193b-5p and occludin was identified. The loss in endothelial integrity was rescued when miR-193b-5p inhibitor was transfected. Conclusion: miR-193b-5p is a suppressor of occludin. Studying transcriptional changes allows identification of therapeutically relevant mediators for ARDS/ALI treatment.

Severe manifestations of malaria, including cerebral malaria (CM) and respiratory distress, result in approximately three million deaths annually worldwide. Currently, relatively little is known about severe disease pathogenesis. The development and outcome of severe malaria is determined by host-pathogen interactions, a complex interface of genetics and immune responses. Hypothetically, a spectrum of genetic susceptibility and resistance to severe disease exists within the host population, and malaria infection results in diverse host and parasite responses that impact disease outcome. The aim of this study was to identify differential host and parasite responses in a murine model of severe malaria, Plasmodium berghei ANKA (PbA), in CM-susceptible and CM-resistant mice; and to analyze host genetics in patients with severe disease due to Plasmodium falciparum. In vivo, expression microarray analysis showed that, in malaria target organs, differential responses were related to immune response – primarily interferon and complement pathways – and apoptosis. Histopathological examination of the brain confirmed an increased prevalence of apoptosis in CM-susceptible mice. Further examination of the role of complement in CM-susceptibility determined that early complement 5 (C5) activation conferred susceptibility to CM, and that C5 deficiency conferred resistance, which could be recapitulated by antibody blockade of activated C5 or its receptor in susceptible mice. Additionally, single nucleotide polymorphism (SNP) studies identified that complement receptor 1 SNPs were associated with disease severity in patients with P. falciparum malaria. PbA parasites displayed a unique transcriptional signature in each tissue examined (brain, liver, spleen and lung), showed differential gene expression between CM-resistant and susceptible hosts, and were most prominent in lung tissue. Closer examination of lung involvement in PbA infection revealed that PbA-infected C57BL/6 mice develop acute lung injury (ALI), defined by disruption of the alveolar-capillary membrane barrier. ALI susceptibility did not correlate with CM susceptibility, but was influenced by peripheral parasite burden and CD36-mediated parasite sequestration in the lung. PbA provides a clinically relevant experimental model for CM and ALI, through which important disease mechanisms can be identified and modulated. Ideally, the use of such models aids in the discovery of disease biomarkers and novel therapeutic strategies, which may be applied to human severe and cerebral malaria.

XB130 is a relatively unstudied novel signal adaptor protein. In the first phase of this study, an in silico search for proteins related to XB130 was conducted. Two other proteins (AFAP and AFAP1L1) were found to have a significant similarity to XB130 and were compared in detail. After an analysis of these three proteins, it was proposed that they are members of a novel protein family, termed the “AFAP family of signal adaptor proteins”. XB130 has previously been found to regulate cell cycle progression, death, and migration in lung epithelial cells. It was therefore hypothesized that XB130 is protective in acute lung injury (ALI) and important for facilitating repair after injury. XB130 was found to be differentially regulated in ALI depending on the initial insult. Engineering XB130 transgenic mice to further characterize the role of XB130 in lung injury/regeneration revealed that this protein could be essential for early embryo development.

Severe manifestations of malaria, including cerebral malaria (CM) and respiratory distress, result in approximately three million deaths annually worldwide. Currently, relatively little is known about severe disease pathogenesis. The development and outcome of severe malaria is determined by host-pathogen interactions, a complex interface of genetics and immune responses. Hypothetically, a spectrum of genetic susceptibility and resistance to severe disease exists within the host population, and malaria infection results in diverse host and parasite responses that impact disease outcome. The aim of this study was to identify differential host and parasite responses in a murine model of severe malaria, Plasmodium berghei ANKA (PbA), in CM-susceptible and CM-resistant mice; and to analyze host genetics in patients with severe disease due to Plasmodium falciparum. In vivo, expression microarray analysis showed that, in malaria target organs, differential responses were related to immune response – primarily interferon and complement pathways – and apoptosis. Histopathological examination of the brain confirmed an increased prevalence of apoptosis in CM-susceptible mice. Further examination of the role of complement in CM-susceptibility determined that early complement 5 (C5) activation conferred susceptibility to CM, and that C5 deficiency conferred resistance, which could be recapitulated by antibody blockade of activated C5 or its receptor in susceptible mice. Additionally, single nucleotide polymorphism (SNP) studies identified that complement receptor 1 SNPs were associated with disease severity in patients with P. falciparum malaria. PbA parasites displayed a unique transcriptional signature in each tissue examined (brain, liver, spleen and lung), showed differential gene expression between CM-resistant and susceptible hosts, and were most prominent in lung tissue. Closer examination of lung involvement in PbA infection revealed that PbA-infected C57BL/6 mice develop acute lung injury (ALI), defined by disruption of the alveolar-capillary membrane barrier. ALI susceptibility did not correlate with CM susceptibility, but was influenced by peripheral parasite burden and CD36-mediated parasite sequestration in the lung. PbA provides a clinically relevant experimental model for CM and ALI, through which important disease mechanisms can be identified and modulated. Ideally, the use of such models aids in the discovery of disease biomarkers and novel therapeutic strategies, which may be applied to human severe and cerebral malaria.