Adequate Levels of Hydrogen Peroxide are Necessary to Macrophage Function
A. Brent Carter, M.D.
University of Iowa Hospitals and Clinics

Asbestosis, which is an important cause of pulmonary fibrosis, results from a high occupational exposure to asbestos (minimum fiber dose of 25-100 fibers/ml/year). Although standards regarding handling asbestos have changed, it is estimated that asbestos-associated deaths in the United States may exceed 200,000 by the year 2030. The initial defense-fighting cells in the lung are known as alveolar macrophages. The lungs of patients with asbestosis contain alveolar macrophages that spontaneously release inflammatory mediators, such as tumor necrosis factor-alpha (TNF-O), and reactive oxygen species, especially hydrogen peroxide (H2O2). In fact, asbestos fibers spontaneously catalyze the generation of reactive oxygen species, and studies have shown that proteins obtained from the lung of patients with asbestosis are oxidized. Studies also indicate that inhibition of TNF-O and H2O2 prevent the development of pulmonary fibrosis, which is a form of scarring in the lung. It is also clear that young macrophages that are newly recruited to the lung are primarily responsible for TNF-O production. We have found that adequate steady-state levels of H2O2 are necessary for TNF-O production. These levels of H2O2 are dependent on the activity of peroxide-generating enzymes, such as superoxide dismutase (SOD), and peroxide-removing enzymes, such as catalase and glutathione peroxidase. Thus, it appears that the balance between oxidant-producing enzymes and antioxidant enzymes regulates TNF-O production.

H2O2 is the most important reactive oxygen species involved in inducing inflammation in the lung. Asbestosis is a good disease model to explore the role of H2O2 in inducing inflammation because asbestos is still present in the environment and is known to induce high levels of H2O2. Although cells exposed to asbestos produce TNF-O and H2O2, the mechanism linking H2O2 generation to TNF-O production is not known. We have found that H2O2 is necessary for optimal production of TNF-O after exposure to asbestos and that the source of H2O2 is primarily from the peroxide-generating enzyme Cu,Zn-SOD. We have also found that alveolar macrophages from asbestosis patients have different enzymes that are activated, and these enzymes are known to be important for the development of inflammation. Our data demonstrates that H2O2 regulates the activation of these enzymes, which are known as mitogen-activated protein (MAP) kinases. Since the activity of these enzymes is often controlled by other proteins known as phosphatases, we have found that H2O2 regulates phosphatases that control the activity of MAP kinases. We also plan on comparing MAP kinase and Cu,Zn-SOD activity and H2O2 generation in alveolar macrophages obtained from asbestosis patients to alveolar macrophages obtained from normal volunteers. We have found that Cu,Zn-SOD activity and H2O2 generation is much greater in alveolar macrophages obtained from asbestosis patients than those obtained from normal volunteers. Although the studies in this proposal relate to asbestosis, they are novel studies that also provide important basic clues to understand the development of inflammation in the lung after exposure to an environmental agent.

Blocking Influenza Virus Replication May Help Protect Against Spread Of "Bird Flu"
Stacey Schultz-Cherry, PhD
University of Wisconsin, Madison, WI

Regulation Of Influenza Virus Replication By p53: Part Of The Antiviral Response? Because the influenza virus changes every year, a new influenza vaccine must be developed every year. Scientists must first identify the viral strain that appears to be prevalent, and then develop a vaccine targeted to that strain. If scientists could block replication of the virus, however, they could develop a vaccine that attacks all strains of the virus much earlier in the process. These researchers are studying the interaction of influenza virus with human lung cells. They have identified a specific cellular protein that inhibits the growth of all influenza viruses tested so far. They are now looking at how the cellular protein regulates viral growth, and how the viral proteins block its function. They hope to use this knowledge to look at dangerous "bird flu" viruses that have been infecting and killing people in Southeast Asia. There are no approved vaccines for these avian influenza viruses, which in 2004 had a 60 percent mortality rate in humans, and are resistant to a common anti-influenza drug. These researchers hope that their work will contribute to solutions to protect against the potentially worldwide spread of avian influenza viruses.

Developing New Drugs To Treat Drug-Resistant Tuberculosis
Michael G. Thomas, PhD
University of Wisconsin, Madison, WI

Deciphering And Manipulating Capreomycin Biosynthesis To Generate New Drugs Against Multidrug-Resistant Tuberculosis. As more strains of Mycobacterium tuberculosis become resistant to first-line antibiotics, there is an increased reliance on second-line drugs to successfully treat multi-drugresistant tuberculosis infections. The capreomycins (CAPs) are a family of second-line drugs that are essential components of the drug regimen used to treat these multi-drug-resistant infections. As the use of these antibiotics becomes more frequent, it is anticipated that resistance to the CAPs will also increase. These researchers aim to develop new CAP derivatives by genetically engineering the biosynthetic machinery that produces these antibiotics in nature. Using this approach, new CAP derivatives will be generated that regain antibacterial activity against resistant strains, which will have enormous potential in extending the lifetime of these antibiotics. Additionally these researchers aim to generate new CAP derivatives that will also have reduced side effects for the patient.

Finding a Way to Help the Immune System Fight off Bacterial Infections After Influenza
Respiratory Dendritic Cells: Cell Migration and Induction of Adaptive Immunity During Virus Infections.
Kevin Legge, Ph.D.
University of Iowa Hospitals and Clinics

The lungs are routinely exposed to foreign pathogens such as bacteria and viruses in the air that we breathe. Often our immune system halts these pathogens before significant infections can occur. However when these pathogens do establish an infection, the immune system must kick in to fight it. In the lungs, respiratory dendritic cells (rDC) are thought to be responsible for inducing this immune system response. The researchers have found that following influenza infections, rDC rapidly migrate from the lungs to the lymph nodes and induce an immune response that is specific to influenza. But within 24 hours after influenza virus infection, rDC halt their migration. This halt is of particular concern following respiratory virus infections, where concurrent or new bacterial infections are common. The researchers will study the way in which rDC migration is stopped following influenza virus infections. They will then determine if manipulating this mechanism can restore rDC migration, thus helping the immune system fight off secondary infections. By boosting the immune system’s response to bacterial infections, complications ranging from otitis media and pneumonia in children to potentially deadly pneumonia in elderly adults might be avoided.

Gene-specific silencing for lung cancer therapy
Kounosuke Watabe, Ph.D.
Southern Illinois University

Lung cancer is the most prevalent malignancy worldwide. It has been estimated that 80% of lung cancer deaths among men and 75% of lung cancer deaths among women are attributable to smoking . The most important cause of the low cure rate in lung cancer is the high frequency of metastatic spread prior to diagnosis. Once metastases occur, patients cannot be cured by currently available therapies. Therefore, development of alternative treatment methods is urgently needed. Fatty acid synthase (FAS) has been found to be expressed specifically in a variety of tumors including lung cancer and hence considered to be an ideal target for chemotherapy. The goal of this project is to examine a possibility of using a small piece of RNA (called siRNA) which can "silence" the FAS gene as a therapeutic drug. In order to use this siRNA as a specific tumor drug, we will integrate this sequence into the adenovirus genome and administer this virus through inhalation to an animal model of lung cancer. We believe that the adenovirus containing siRNA to the fatty acid synthase gene specifically suppresses the expression of this gene and causes tumor cell death. If the result of the experiment is promising, we can eventually use this agent in a clinical trial. We believe that this unique design for the therapeutical approach to lung cancer will eventually yield an effective and non-toxic anti-cancer drug.

How Can Lung Cells That Cause Scarring Be Turned Off?
Deanna Diebold, MD
University of Minnesota, Minneapolis, MN

Translational Control Of Collagen Matrix Contraction-Triggered Fibroblast Apoptosis. Idiopathic pulmonary fibrosis (IPF) involves progressive scarring of the lung tissue, causing respiratory failure and ultimately death. At the present time, little is known about the cause of IPF and there are few available treatments. The disease results from the abnormal proliferation of connective tissue cells called fibroblasts. Normally, when the lung is injured, fibroblasts are recruited to the site of injury, but once healing is completed these cells die off. However in IPF, fibroblasts continue to proliferate, leading to lung scarring. The researchers will study fibroblasts to obtain information about how these cells are involved in normal healing vs. scarring. Understanding the regulatory factors that determine whether fibroblasts persist abnormally or die off during lung repair after injury may lead to new therapeutic approaches for treating IPF.

Improving the Treatment of Asthma
Malcolm Blumenthal, MD
University of Minnesota
ACRC Principal Investigator

The Asthma Clinical Research Centers (ACRC) Network, sponsored by the American Lung Association, conducts large clinical trials that provide vital information about caring for people who have asthma. The Network comprises 20 clinical centers and a data coordinating center, making it the largest of its kind. Its unique focus on large numbers of patients differentiates it from current federally funded and commercial research, and provides practical information about asthma care that has direct benefits for patients. The ACRC Network is currently conducting the following studies.

Improving the Treatment of Asthma
Lewis Smith, MD
Northwestern Center for Clinical Research
ACRC Principal Investigator

The Asthma Clinical Research Centers (ACRC) Network, sponsored by the American Lung Association, conducts large clinical trials that provide vital information about caring for people who have asthma. The Network comprises 20 clinical centers and a data coordinating center, making it the largest of its kind. Its unique focus on large numbers of patients differentiates it from current federally funded and commercial research, and provides practical information about asthma care that has direct benefits for patients. The ACRC Network is currently conducting the following studies.

Inhibiting Enzyme May Help Prevent Lung Transplant Rejection
Andrés Jaramillo, PhD
Rush University Medical Center, Chicago, IL

Matrix Metalloprotainase-9 In Experimental Bronchiolitis Obliterans. The long-term survival of lung transplant recipients is limited by the development of extensive scarring tissue (fibrosis) of the small airways, a condition known as bronchiolitis obliterans (BO) that represents chronic lung transplant rejection. However, current treatments are not effective in preventing BO, and the origin and development of the disease are not well understood. A growing body of evidence has indicated that BO is induced by the immune system’s inflammatory response to the transplanted lung. The researchers are studying the role of the immune response in the pathogenesis of BO. Using a mouse model of tracheal transplantation, the researchers have found that levels of an enzyme known as matrix metalloproteinase-9 are significantly elevated during the development of BO, and that treatment with inhibitors of this enzyme hampers the development of the disease. The researchers will further study the specific role of this enzyme in the immune response that develops after lung transplantation using genetically engineered mice deficient in this enzyme. The results of this study will provide crucial information about the pathogenesis of BO and may potentially lead to therapeutic alternatives for its treatment and/or prevention.

Inhibiting Formation Of Bacterial Structures In Lungs Of Patients With Cystic Fibrosis
Timothy D. Starner, MD
University of Iowa, Iowa City, IA

Nontypeable Haemophilus Influenzae Biofilm Formation On Airway Epithelia. Nontypeable Haemophilus influenzae (NTHi) is one of the first bacteria to infect the lower airways of cystic fibrosis patients. NTHi infections may pave the way for other bacteria that cause extensive disease in cystic fibrosis. It has been shown in the lungs of cystic fibrosis patients that bacteria grow in a slime-like layer, called a biofilm. These biofilms are more resistant to antibiotics and to the body’s defenses. The researchers will study biofilm formation of NTHi. Understanding factors that can inhibit biofilm formation of NTHi may lead to new areas of research in cystic fibrosis or identify new targets for treatment.

Mechanism of Urokinase Plasminogen Activator Activation of Airway Eosinophil Function in Asthma
Julie Sedgwick, Ph.D.
University of Wisconsin-Madison

While a great deal has been learned about the mechanisms of asthma, there are still many poorly understood aspects of this lung disease. Eosinophils are potentially destructive cells that promote airway inflammation in asthma, but the mechanism by which they invade the airways of asthmatics and become activated has not been well defined. The researchers will look at how eosinophils are affected by substances called urokinase plasminogen activator receptors (uPAR), and study the role of increased eosinophils in the airways of people with allergic disease and asthma. This information will be critical to therapeutic strategies aimed at this cell.

Neutrophil-Endothelial Cell Interactions Elicited by S. pneumoniae in the Lung
Jessica G. Moreland, M.D.
University of Iowa Hospitals and Clinics

Streptococcus pneumoniae is the most common cause of bacterial pneumonia in both adults and children. This type of pneumonia can be very severe and may lead to bloodstream infection and death in a significant number of cases. One of the earliest events in our immune system’s response to pneumonia is the movement of white blood cells from the bloodstream to the lung. The principal long-term goal of this research project is to better understand some of the early events in the host response to bacterial pneumonia that initiate the movement of white blood cells from within the bloodstream to the lung, and may enhance the ability of the white blood cell to attack and kill the bacteria that is causing pneumonia. We have selected Streptococcus pneumoniae as the bacteria for our proposed studies for three reasons: 1) S. pneumoniae is the most common cause of bacterial pneumonia in adults and children, 2) this bacteria frequently exits the lung and moves into the bloodstream indicating that it directly contacts the endothelial cells or cells that line the blood vessels of the lung, and 3) pneumonia caused by this bacteria is characterized by large numbers of white blood cells moving from the bloodstream into the lung and causing severe inflammation .

Our specific focus involves investigation of the interaction of bacteria with both the human lung endothelial cells that line our blood vessels and human neutrophils. The overall hypothesis of this proposal is that Streptococcus pneumoniae elicits specific endothelial cell responses that directly participate in recruitment of white blood cells (neutrophils) from the vascular space to the lung during the onset of bacterial pneumonia. We propose that the white blood cells are activated by the process of migrating out of the bloodstream to the lung. To explore these questions, we plan to characterize the interaction between intact, live S. pneumoniae and pulmonary microvascular endothelial cells. Furthermore, we will define functional changes in the white blood cell (neutrophil) following migration across the endothelial cells that may enhance the ability of these white blood cells to destroy bacteria (e.g. S. pneumoniae) and/or surrounding host tissues. These questions are studied using a cell culture system to model events that occur in the body.

The Regulation of Myofibroblast Development and Apoptosis
Eun Jun Yun, Ph.D.
University of California

The purpose of this study is to address the role of myofibroblast development and death in distal lung morphogenesis. Distal airway (alveoli) formation is complex process requiring communication between various cells and its mechanism is largely unknown. Especially, the mechanisms that regulate myofibroblast development are not fully understood to date. Disrupted lung myofibroblast development and maintenance of alveolar structure may lead to pulmonary disorders such as bronchopulmonary dysplasia, fibrosis and emphysema. Therefore, it is important to understand the origin and development of this cell type to gain insights into both developmental and pathological processes. We believe that the growth factors that regulate compensatory growth will play a similar role in regulating alveolar septation in the newborn and will lead to new treatments for emphysema. Of note, the fate of tissue fibrosis is in part regulated via a balance between myofibroblast survivals vs. death. Our hypothesis is that balance between myofibroblast development and reducing its number is necessary for both alveolar septae formation in developing lungs and maintenance of alveoli structure in adult lungs. Based on results and findings from our previous studies, we hypothesized that the functions of growth factors HGF (Hepatocyte growth factor) and VEGF (Vascular endothelial growth factor) are closely associated in reciprocal interactions and probably regulate this balance.

We use genetically modified mice to study the function of these growth factors on myofibroblast development. Preliminary data show two interesting distinct phenotype with the loss of VEGF during postnatal period. If we decrease VEGF expression during early postnatal period, we find reduced number of myofibroblast and elastin. If we decrease VEGF expression during late postnatal period, we see lung destruction like emphysema. PECAM staining indicated abnormal distribution of pulmonary capillaries in the enlarged airways in these transgenic mice. We are currently examining for growth factors regulating these processes.

The results obtained from these studies will further our understanding of the mechanisms of lung formation and of the development of lung fibrosis and may provide novel targets for the development of new antifibrotic therapies.