Respiratory syncytial virus (RSV) is the primary viral pathogen responsible for lower respiratory tract disease in neonates and young children worldwide. By the age of two, virtually all children have been infected with RSV, and approximately 40% of them develop lower respiratory tract infections. In addition to acute morbidity, an association between RSV infection in early childhood and later development of recurrent wheezing and airway hyperresponsiveness (AHR) has been repeatedly demonstrated.<p>In this work we established a method for propagating pneumonia virus of mice (PVM) in a baby hamster kidney-21 (BHK-21) cell line. We also modified the standard plaque assay method and established a reliable and, most importantly, reproducible way to quantitate PVM. In our work we used PVM strain 15 to successfully establish an in vivo animal model for RSV disease in Balb/c and C57/Bl mice. Different susceptibility/resistance patterns to a pathogen exist for different mouse strains. In the case of Balb/c and C57/Bl mice, these patterns are well characterized for several pathogens including Leishmania major and adenovirus type 1. Our comparative study demonstrated clear differences in susceptibility to PVM strain 15 infection between Balb/c and C57/Bl mice; Balb/c mice being more susceptible.<p> In peripheral sites, dendritic cells (DCs) serve as sentinel cells that take up and process antigens. Numerous studies revealed that certain pathogens stimulate changes in DC phenotypic characteristics and thus contribute to functional alterations that lead to inappropriate T cell activation and disease augmentation. To examine effects of PVM on DCs, we infected bone marrow dendritic cells (BM-DCs) derived from both mouse strains with PVM, and evaluated their phenotypic and functional characteristics 24 hours post infection. Under these experimental conditions, PVM infected BM-DCs did not show a significant increase in the expression of costimulatory and major histocompatibility complex class II (MHC II) molecules compared to uninfected controls. Furthermore, there were no changes in the ability of PVM-infected DCs to take up soluble antigen. The production of IL-12p70, the pivotal cytokine in the development of a Th1-type response, by the PVM-infected BM-DCs was not significantly different from uninfected cells. In addition, there was no significant impact of PVM infection on the ability of DCs to induce naïve T cell proliferation.

The objectives of this project were: 1) to determine if live animal lung biopsy could be used to characterize early pathologic changes in the bovine lung associated with bovine respiratory disease (BRD), 2) determine if specific infectious respiratory pathogens can be identified in association with early pathological changes, and 3) determine whether pulmonary pathology characterized by live animal lung biopsy at arrival and at the time of initial BRD diagnosis was associated with health and production outcomes of feedlot steers in a commercial feedlot.<p> A live animal percutaneous lung biopsy technique was developed to obtain a lung sample from the right middle lung lobe in intercostal space (ICS) 4 using a Bard® Magnum® reusable biopsy instrument and a modified 4-mm (8g) biopsy needle. The lung biopsy procedure was limited to 2 attempts per biopsy time. In the technique development, 34 animals chronically affected with BRD were utilized, 20 animals in the preliminary development followed by 14 additional animals in a commercial feedlot setting. The technique resulted in 1 fatality of 34 steers (2.9%) and lung parenchyma was harvested in 19 of 34 steers (55.9%) chronically affected with BRD. In addition, in the commercial feedlot setting this procedure was determined to take about 20 minutes per animal.<p> The final study was performed on one hundred feedlot steers considered at high risk of developing BRD from twenty pens within a commercial feedlot. Study animals were enrolled in three different groups: sick on arrival (ARR-SA) consisting of 27 study animals and 13 matched control animals; pen pulls with no fever (PP-NF) consisting of 14 study animals and matched 7 controls; and pen pulls with an undifferentiated fever (PP-UF) consisting of 26 study animals and 13 matched controls. Live animal percutaneous lung biopsies were collected from the right middle lung lobe at 3 different times within the first 30 days of the feeding period, about 2 weeks apart. All samples were histopathologically evaluated and were assessed for the presence of <i>Mycoplasma bovis</i>, <i>Mannheimia haemolytica</i>, Histophilus somni and bovine viral diarrhea virus with immunohistochemistry.<p> A total of 295 lung biopsies were performed yielding 210 (71.2%) lung samples that were sufficient for histopathological evaluation. A histopathology score was awarded to each biopsy based on certain histopathological lesions being present. Only 20 lung biopsy samples from 19 animals received a histopathology score (ie, pulmonary lesions were present) with the most common score being a 1 (maximum score is 20). There were too few lung biopsy samples with a histopathology score to reveal any association with subsequent health events.<p> Immunohistochemistry (IHC) was performed on all lung biopsies recovered yielding one lung sample to be positive for both <i>Mannheimia haemolytica</i> and <i>Mycoplasma bovis</i> from the PP-UF group. There were too few positive samples to reveal any association between IHC and histopathology score.<p> A post mortem evaluation was performed by a study veterinarian on all study animals who died or were humanely euthanized due to poor treatment response. In this study only 4 steers died or were euthanized due to poor treatment response and 3 control steers were humanely euthanized. There were too few animals to reveal any association between histopathology score and post mortem diagnosis.<p> On entry into the feedlot, weights between ARR-SA and the PP-UF and PP-NF groups were significantly different (p<0.05). This is likely an effect of the different processing groups of cattle. At study allocation, the body weights of ARR-SA and PP-UF, PP-UF and their matched controls, and PP-NF and their matched controls were also significantly different (p<0.05). This is likely due to the PP-UF and PP-NF groups experiencing illness for a longer period of time resulting in greater weight loss than the ARR-SA animals as well as the control animals, who were not clinically sick.<p> The live lung biopsy procedure utilized in this study did not appear to cause any long lasting adverse effects as the BRD case fatality rates from the study animals were comparable to the overall case fatality rates reported by the feedlot for fall placed calves. In fact, the study animals experienced a decreased fatality rate compared to the feedlots overall fatality rate. This may be due to the study animals inadvertently being monitored more closely as the pen checkers were aware of and participating in the study. On post mortem evaluation there was no evidence of adhesions at the biopsy site. This procedure was performed on 134 feedlot steers resulting in only 2 acute deaths as a direct result of the live animal percutaneous lung biopsy procedure.<p> The results of this study indicate that live animal, percutaneous lung biopsy can be performed safely on feedlot steers in a commercial feedlot with few clinical side effects. In this study there were only 2 fatalities in 134 steers (1.5%) due to the biopsy procedure or 2 fatalities per 349 sampling times (0.6%) This technique did not prove useful either as a diagnostic tool for the determination of early lung pathology in BRD or as prognostic indicator for health and production outcomes. However, this lung biopsy technique may be a useful diagnostic tool for chronic pneumonia assessment.

Radiation therapy for lung cancer and cancers of the upper thorax is limited by side effects to normal tissue of the lung. An understanding of mechanisms leading to radiation induced lung damage is essential to developing protective agents. In this thesis an anti-oxidant and anti-inflammatory agent Genistein was investigated for its potential to affect DNA damage, tissue inflammation, functional deficits and survival. We hypothesized that chronic oxidative stress and the subsequent inflammatory response play a key role in the development of major lung complications, radiation pneumonitis and fibrosis. If side effects of radiation could be reduced, then larger doses could be delivered to the tumor with a better chance of eradicating the disease.

The alveoli of mammalian lungs are covered in a thin lipid film referred to as pulmonary surfactant. The primary purpose of pulmonary surfactant is to reduce the surface tension of the air/liquid interface allowing breathing with minimal effort required. We investigated the effect of addition of cholesterol and amyloid-β peptide on structure and function of Bovine Lung Extract Surfactant (BLES) and model lipid films. In our first experiment, we have demonstrated the effect of amyloid-β and cholesterol on lipid films of DPPC, DPPC-DOPG and BLES. We saw that cholesterol inhibits multilayer formation in all monolayers. Amyloid-β increases multilayer formation in DPPC and DPPC-DOPG, but reduced multilayer formation in BLES. When cholesterol and amyloid-β is added to BLES, 1% amyloid-β is inconsequential, whereas 10% amyloid-β allows BLES to regain some of its surfactant function. In our second experiment, we observed that for bothanionic DOPG and cationic DOTAP films which are in the fluid phase, amyloid-β interacts with the bilayer much quicker than in zwitterionic DPPC which is in the gel phase. Approaching 24 hours, we see small fibrils form on the bilayer, but these fibrils are considerably smaller than those formed when amyloid-β is incubated in solution. For fluid phase bilayer membrane, disruption is also observed. We investigated the effect of addition of cholesterol and amyloid-β peptide on structure and function of Bovine Lung Extract Surfactant (pulmonary surfactant BLES) and model lipid films. In our first experiment, we have demonstrated the effect of amyloid-β and cholesterol on lipid films of DPPC, DPPC-DOPG and BLES. We saw that cholesterol inhibits multilayer formation in all monolayers. Amyloid-β increases multilayer formation in DPPC and DPPC-DOPG, but reduced multilayer formation in BLES. When cholesterol and amyloid-β is added to BLES, 1% amyloid-β is in consequential, whereas 10% amyloid-β allows BLES to regain some of its surfactant function. In our second experiment, we observed that in anionic DOPG films, amyloid-β inserts into the bilayer much quicker than in zwitterionic DPPC. Approaching 24 hours, we see small fibrils form in the bilayer, but these fibrils are considerably smaller than those formed when amyloid-β is incubated in solution.

The alveoli of mammalian lungs are covered in a thin lipid film referred to as pulmonary surfactant. The primary purpose of pulmonary surfactant is to reduce the surface tension of the air/liquid interface allowing breathing with minimal effort required. We investigated the effect of addition of cholesterol and amyloid-β peptide on structure and function of Bovine Lung Extract Surfactant (BLES) and model lipid films. In our first experiment, we have demonstrated the effect of amyloid-β and cholesterol on lipid films of DPPC, DPPC-DOPG and BLES. We saw that cholesterol inhibits multilayer formation in all monolayers. Amyloid-β increases multilayer formation in DPPC and DPPC-DOPG, but reduced multilayer formation in BLES. When cholesterol and amyloid-β is added to BLES, 1% amyloid-β is inconsequential, whereas 10% amyloid-β allows BLES to regain some of its surfactant function. In our second experiment, we observed that for bothanionic DOPG and cationic DOTAP films which are in the fluid phase, amyloid-β interacts with the bilayer much quicker than in zwitterionic DPPC which is in the gel phase. Approaching 24 hours, we see small fibrils form on the bilayer, but these fibrils are considerably smaller than those formed when amyloid-β is incubated in solution. For fluid phase bilayer membrane, disruption is also observed. We investigated the effect of addition of cholesterol and amyloid-β peptide on structure and function of Bovine Lung Extract Surfactant (pulmonary surfactant BLES) and model lipid films. In our first experiment, we have demonstrated the effect of amyloid-β and cholesterol on lipid films of DPPC, DPPC-DOPG and BLES. We saw that cholesterol inhibits multilayer formation in all monolayers. Amyloid-β increases multilayer formation in DPPC and DPPC-DOPG, but reduced multilayer formation in BLES. When cholesterol and amyloid-β is added to BLES, 1% amyloid-β is in consequential, whereas 10% amyloid-β allows BLES to regain some of its surfactant function. In our second experiment, we observed that in anionic DOPG films, amyloid-β inserts into the bilayer much quicker than in zwitterionic DPPC. Approaching 24 hours, we see small fibrils form in the bilayer, but these fibrils are considerably smaller than those formed when amyloid-β is incubated in solution.

Respiratory syncytial virus (RSV) is the primary viral pathogen responsible for lower respiratory tract disease in neonates and young children worldwide. By the age of two, virtually all children have been infected with RSV, and approximately 40% of them develop lower respiratory tract infections. In addition to acute morbidity, an association between RSV infection in early childhood and later development of recurrent wheezing and airway hyperresponsiveness (AHR) has been repeatedly demonstrated.<p>In this work we established a method for propagating pneumonia virus of mice (PVM) in a baby hamster kidney-21 (BHK-21) cell line. We also modified the standard plaque assay method and established a reliable and, most importantly, reproducible way to quantitate PVM. In our work we used PVM strain 15 to successfully establish an in vivo animal model for RSV disease in Balb/c and C57/Bl mice. Different susceptibility/resistance patterns to a pathogen exist for different mouse strains. In the case of Balb/c and C57/Bl mice, these patterns are well characterized for several pathogens including Leishmania major and adenovirus type 1. Our comparative study demonstrated clear differences in susceptibility to PVM strain 15 infection between Balb/c and C57/Bl mice; Balb/c mice being more susceptible.<p> In peripheral sites, dendritic cells (DCs) serve as sentinel cells that take up and process antigens. Numerous studies revealed that certain pathogens stimulate changes in DC phenotypic characteristics and thus contribute to functional alterations that lead to inappropriate T cell activation and disease augmentation. To examine effects of PVM on DCs, we infected bone marrow dendritic cells (BM-DCs) derived from both mouse strains with PVM, and evaluated their phenotypic and functional characteristics 24 hours post infection. Under these experimental conditions, PVM infected BM-DCs did not show a significant increase in the expression of costimulatory and major histocompatibility complex class II (MHC II) molecules compared to uninfected controls. Furthermore, there were no changes in the ability of PVM-infected DCs to take up soluble antigen. The production of IL-12p70, the pivotal cytokine in the development of a Th1-type response, by the PVM-infected BM-DCs was not significantly different from uninfected cells. In addition, there was no significant impact of PVM infection on the ability of DCs to induce naïve T cell proliferation.

Glu-Leu-Arg (ELR)-CXC chemokine-mediated neutrophil migration and activation plays a key role in many inflammatory diseases. Dysregulated neutrophil activation often leads to inflammatory responses such as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS).<p> Previously, we generated a bovine drug (i.e., bovine CXCL8(3-74)K11R/G31P, bG31P) by mutating the first two amino acids at the beginning of the N-terminus of bovine CXCL8/IL-8 and later substituting Arg for Lys11 and Pro for Gly31. Bovine G31P was shown to be a highly effective ELR-CXC chemokine and neutrophil antagonist in cattle & guinea pigs, but a human equivalent thereof would be of significantly more use in human medicine. Published studies on the structure and function of human CXCL8 suggest that human CXCL8(3-72)K11R/G31P (i.e., hG31P) would not be a particularly effective chemokine antagonist. Thus, development of a humanized form of bG31P became a primary goal. I first examined the effect of wholesale ligation of the carboxy half of hCXCL8 onto the amino half of bG31P and generated a human-bovine chimeric G31P (hbG31P; i.e., bCXCL8(3-44)K11R/G31P-hCXCL8(45-72)). I also made substitutions at each remaining human-discrepant amino acid (i.e., T3K, H13Y, T15K, E35A, and S37T) within the 5 half of the hbG31P cDNA. The results showed that hbG31P and its analogues blocked CXCL8-induced human neutrophil chemotactic responses, reactive oxygen intermediate (ROI) release, and intracellular calcium flux. Humanized bovine G31P was also shown to significantly block pulmonary neutrophilic pathology in a guinea pig model of airway endotoxemia.<p> As bG31P, hbG31P and its further humanized forms showed essentially equivalent ELR-CXC chemokine antagonist activity, Dr. Fang Li, Ms Jennifer Town and I then generated a fully human form of bG31P, hG31P. <i>In vitro</i>, hG31P was shown to effectively inhibit CXCL-1-, -5-, and -8-induced neutrophil chemotactic responses, intracellular Ca2+ flux, and ROI release. Human G31P also desensitized heterologous G protein-coupled receptors (GPCR) including bacterial peptides (e.g., N-formyl-methionine-leucine-phenylalanine, fMLP), anaphylatoxin (e.g., complement 5a, C5a), lipid mediators (e.g., leukotriene B4, LTB4; platelet-activating factor, PAF) receptors. Moreover, hG31P, in a dose-dependent manner suppressed CXCL1 and CXCL8 expression by LPS-challenged airway epithelial cells and reversed the anti-apoptotic influence of ELR-CXC chemokines on neutrophils. <i>In vivo</i>, hG31P was significantly effective in blocking the pathology associated with airway endotoxemia, aspiration pneumonia, and intestinal ischemia and reperfusion injury, including neutrophil recruitment (70-95% reduction) into, and activation within, the airways or gut, chemokine or cytokine expression, and pulmonary vascular complications. The blockade of neutrophil recruitment by hG31P in aspiration pneumonia animals did not increase airway bacterial growth. The G31P treatment was protective in both mesenteric (i.e., local) and remote organ injury. These findings suggest that hG31P is not only a potent neutrophil antagonist, but an effective blocker of other inflammatory responses. These comprehensive anti-inflammatory effects indicate that hG31P could potentially provide a viable therapeutic approach for inflammatory diseases such as ALI /ARDS.

The objectives of this project were: 1) to determine if live animal lung biopsy could be used to characterize early pathologic changes in the bovine lung associated with bovine respiratory disease (BRD), 2) determine if specific infectious respiratory pathogens can be identified in association with early pathological changes, and 3) determine whether pulmonary pathology characterized by live animal lung biopsy at arrival and at the time of initial BRD diagnosis was associated with health and production outcomes of feedlot steers in a commercial feedlot.<p> A live animal percutaneous lung biopsy technique was developed to obtain a lung sample from the right middle lung lobe in intercostal space (ICS) 4 using a Bard® Magnum® reusable biopsy instrument and a modified 4-mm (8g) biopsy needle. The lung biopsy procedure was limited to 2 attempts per biopsy time. In the technique development, 34 animals chronically affected with BRD were utilized, 20 animals in the preliminary development followed by 14 additional animals in a commercial feedlot setting. The technique resulted in 1 fatality of 34 steers (2.9%) and lung parenchyma was harvested in 19 of 34 steers (55.9%) chronically affected with BRD. In addition, in the commercial feedlot setting this procedure was determined to take about 20 minutes per animal.<p> The final study was performed on one hundred feedlot steers considered at high risk of developing BRD from twenty pens within a commercial feedlot. Study animals were enrolled in three different groups: sick on arrival (ARR-SA) consisting of 27 study animals and 13 matched control animals; pen pulls with no fever (PP-NF) consisting of 14 study animals and matched 7 controls; and pen pulls with an undifferentiated fever (PP-UF) consisting of 26 study animals and 13 matched controls. Live animal percutaneous lung biopsies were collected from the right middle lung lobe at 3 different times within the first 30 days of the feeding period, about 2 weeks apart. All samples were histopathologically evaluated and were assessed for the presence of <i>Mycoplasma bovis</i>, <i>Mannheimia haemolytica</i>, Histophilus somni and bovine viral diarrhea virus with immunohistochemistry.<p> A total of 295 lung biopsies were performed yielding 210 (71.2%) lung samples that were sufficient for histopathological evaluation. A histopathology score was awarded to each biopsy based on certain histopathological lesions being present. Only 20 lung biopsy samples from 19 animals received a histopathology score (ie, pulmonary lesions were present) with the most common score being a 1 (maximum score is 20). There were too few lung biopsy samples with a histopathology score to reveal any association with subsequent health events.<p> Immunohistochemistry (IHC) was performed on all lung biopsies recovered yielding one lung sample to be positive for both <i>Mannheimia haemolytica</i> and <i>Mycoplasma bovis</i> from the PP-UF group. There were too few positive samples to reveal any association between IHC and histopathology score.<p> A post mortem evaluation was performed by a study veterinarian on all study animals who died or were humanely euthanized due to poor treatment response. In this study only 4 steers died or were euthanized due to poor treatment response and 3 control steers were humanely euthanized. There were too few animals to reveal any association between histopathology score and post mortem diagnosis.<p> On entry into the feedlot, weights between ARR-SA and the PP-UF and PP-NF groups were significantly different (p<0.05). This is likely an effect of the different processing groups of cattle. At study allocation, the body weights of ARR-SA and PP-UF, PP-UF and their matched controls, and PP-NF and their matched controls were also significantly different (p<0.05). This is likely due to the PP-UF and PP-NF groups experiencing illness for a longer period of time resulting in greater weight loss than the ARR-SA animals as well as the control animals, who were not clinically sick.<p> The live lung biopsy procedure utilized in this study did not appear to cause any long lasting adverse effects as the BRD case fatality rates from the study animals were comparable to the overall case fatality rates reported by the feedlot for fall placed calves. In fact, the study animals experienced a decreased fatality rate compared to the feedlots overall fatality rate. This may be due to the study animals inadvertently being monitored more closely as the pen checkers were aware of and participating in the study. On post mortem evaluation there was no evidence of adhesions at the biopsy site. This procedure was performed on 134 feedlot steers resulting in only 2 acute deaths as a direct result of the live animal percutaneous lung biopsy procedure.<p> The results of this study indicate that live animal, percutaneous lung biopsy can be performed safely on feedlot steers in a commercial feedlot with few clinical side effects. In this study there were only 2 fatalities in 134 steers (1.5%) due to the biopsy procedure or 2 fatalities per 349 sampling times (0.6%) This technique did not prove useful either as a diagnostic tool for the determination of early lung pathology in BRD or as prognostic indicator for health and production outcomes. However, this lung biopsy technique may be a useful diagnostic tool for chronic pneumonia assessment.

In recent years, computer tomography (CT) has become an increasingly important tool in the clinical diagnosis, mainly because of the advent of fast scanning techniques and high spatial resolution of the vision hardware. In addition to the detailed information of morphology, functional CT also gives the physiologic information, such as perfusion. It can help doctors to make better decision. Our goal in this paper is to evaluate lung perfusion by comparing pre and post contrast-enhanced CT images. After the contrast agent is injected, it flows with blood stream and causes the temporal changes in CT values. Therefore, we can quantize perfusion values from the changes of CT values between pre and post contrast-enhanced CT images. Then guided by color -coded maps, a quantitative analysis for the assessment of lung perfusion can be performed. As a result, it is easier for observer to determinate the lung perfusion distribution. Moreover, we can use color - coded images to visualize pulmonary embolism and monitor therapeutic efficacy.

The RAF-MEK-ERK is an important signaling pathway that controls cellular proliferation, differentiation and survival. Recent reports indicate that R-RAF is mutated at a high frequency in human cancer. The mutations are clustered in the glycine-rich loop and activation segment which are encoded by exon 11 and exon 15, respectively. Among these mutations, V600E is the most prevalent found in varieties of human cancers, include melanoma and thyroid carcinomas. In this thesis, we analyzed 86 human cancer specimens, including 62 lung cancers and 24 esophageal cancers, for the mutation of exons 11 and 15 by PCR and direct DNA sequencing. However, we can not detect any mutation in these two exons in these clinical samples, these results suggest indicating that BRAF mutation might be rare and analysis of larger sample size is needed to confirmed this conclusion.