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Efficacy of mucolytic treatment in decreasing airway resistance and increasing mucus clearance in a mouse model of asthma

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Title:
Efficacy of mucolytic treatment in decreasing airway resistance and increasing mucus clearance in a mouse model of asthma
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Morgan, Leslie Elizabeth ( author )
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Denver, Colo.
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University of Colorado Denver
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Master's ( Master of integrated sciences)
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University of Colorado Denver
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Integrated Sciences Program, CU Denver
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Integrated sciences

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Asthma ( lcsh )
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bibliography ( marcgt )
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non-fiction ( marcgt )

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Asthma is a serious disease that affects over 25 million people in the United States. Presently, treatment regimens for asthma have focused primarily on controlling smooth muscle contraction (bronchoconstriction) and inflammation. Another feature of asthma, mucus plugging, is prominent in fatal asthma. Mucus hypersecretion is thought to contribute to mild to moderate disease, but its role in non-fatal asthma exacerbations is less clear. This poor understanding of the role of mucus in asthma stems from a lack of knowledge about the production and function of mucin glycoproteins. To this end, recent findings using sensitive molecular and genetic analyses have begun to clarify how mucus affects asthma. Specifically, the polymeric mucin glycoprotein MUC5AC is selectively overproduced in human asthma, and in mice it is required for airway hyperresponsiveness (AHR), a pathophysiological readout of the asthma phenotype. The purpose of my studies was to determine whether a compound that disrupts mucin glycoprotein polymers can prevent AHR. To accomplish this, a mouse model of allergic asthma was created using BALB/cJ wild type (WT) mice challenged with Aspergillus oryzae extract (AOE). Mice were ventilated, and respiratory system resistance (Rrs) was measured at baseline, and in response to methacholine (MCh)-induced smooth muscle contraction and mucin secretion. Rrs increased eight-fold in AOE challenged WT mice compared to unchallenged non-allergic mice. Treatment of AOE challenged WT mice with the mucolytic compound P-3001 disrupted mucin polymers and prevented AHR. Histology showed that occlusion of the airways with mucus was improved in mucolytic treated WT mice. In summary, by demonstrating that a novel mucolytic compound is capable of reversing AHR and mucus plugging in mice via disruption of mucin polymers and increased mobilization of mucus, I have shown that reversing mucus hypersecretion is a potential therapeutic strategy for allergic asthma.
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by Leslie Elizabeth Morgan.

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Full Text
EFFICACY OF MUCOLYTIC TREATMENT IN DECREASING
AIRWAY RESISTANCE AND INCREASING MUCUS CLEARANCE IN A MOUSE MODEL OF ASTHMA by
LESLIE ELIZABETH MORGAN
B.H.S., University of Missouri, 2012
A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Master of Integrated Sciences Integrated Sciences Program
2017


This thesis for the Master of Integrated Sciences degree by Leslie Elizabeth Morgan has been approved for the Integrated Sciences Program by
Christopher Evans, Chair Marc Goal stone Loren Cobb
May 13, 2017
11


Morgan, Leslie Elizabeth (MIS, Integrated Sciences Program)
Efficacy of Mucolytic Treatment in Decreasing Airway Resistance and Increasing Mucus
Clearance in a Mouse Model of Asthma
Thesis directed by Associate Professor Christopher M. Evans
ABSTRACT
Asthma is a serious disease that affects over 25 million people in the United States. Presently, treatment regimens for asthma have focused primarily on controlling smooth muscle contraction (bronchoconstriction) and inflammation. Another feature of asthma, mucus plugging, is prominent in fatal asthma. Mucus hypersecretion is thought to contribute to mild to moderate disease, but its role in non-fatal asthma exacerbations is less clear. This poor understanding of the role of mucus in asthma stems from a lack of knowledge about the production and function of mucin glycoproteins. To this end, recent findings using sensitive molecular and genetic analyses have begun to clarify how mucus affects asthma.
Specifically, the polymeric mucin glycoprotein MUC5AC is selectively overproduced in human asthma, and in mice it is required for airway hyperresponsiveness (AHR), a pathophysiological readout of the asthma phenotype. The purpose of my studies was to determine whether a compound that disrupts mucin glycoprotein polymers can prevent AHR. To accomplish this, a mouse model of allergic asthma was created using BALB/cJ wild type (WT) mice challenged with Aspergillus oryzae extract (AOE). Mice were ventilated, and respiratory system resistance (Rrs) was measured at baseline, and in response to methacholine (MCh)-induced smooth muscle contraction and mucin secretion. Rrs increased eight-fold in AOE challenged WT mice compared to unchallenged non-allergic mice. Treatment of AOE challenged WT mice with the mucolytic compound P-3001 disrupted
m


mucin polymers and prevented AHR. Histology showed that occlusion of the airways with mucus was improved in mucolytic treated WT mice. In summary, by demonstrating that a novel mucolytic compound is capable of reversing AHR and mucus plugging in mice via disruption of mucin polymers and increased mobilization of mucus, I have shown that reversing mucus hypersecretion is a potential therapeutic strategy for allergic asthma.
The form and content of this abstract are approved. I recommend its publication.
Approved: Christopher M. Evans
IV


ACKNOWLEDGEMENTS
The use of animals in this study was authorized under the University of Colorado Denver Institutional Animal Care and Use Committee (IACUC) protocol number:
B-97414(12)1E
This study was conducted with the assistance of the following grants:
Role of Mucin in Lung Homeostasis and Pathophysiology, R01 HL080396, NIH (Evans -PI), 8/1/2009-11/30/2018
MUC5B, a novel therapeutic target for Idiopathic Pulmonary Fibrosis (IPF), UH2 HL123442, NIH (Schwartz PI), 7/1/2014-6/30/19
I would first like to thank Dr. Martin Huber of the Master of Integrated Sciences Program for all of his support and encouragement throughout my time at University of Colorado Denver.
I would also like to thank my thesis committee, Dr. Marc Goalstone and Dr. Loren Cobb.
Profound thanks to the Chair of my committee, Dr. Christopher Evans, who showed me consistent kindness, patience, support, and direction when I needed it. My eternal gratitude to you for affording me this amazing opportunity.
Thanks also to Dr. Adrianne Stefanski for all of her sound advice and guidance with everything from bench work to professional etiquette.
Thanks to all of my coworkers in the Evans lab for your assistance, comradery, and for putting up with all of my shenanigans. Naoko, Vanessa, Rachel, Anna, and to Amanda for listening to all of my woes. Of special note, Dorota Raclawska for being my trainer and go to person for all things lab related.
Ive learned that people will forget what you said, people will forget what you did, but people will never forget how you made them feel -Maya Angel ou
v


DEDICATION
I would like to express my profound gratitude to my husband Dan for providing me with unfailing support and continuous encouragement throughout my years of study and through the process of researching and writing this thesis. This accomplishment would not have been possible without you. A small nod to Oscar for doing what you do.
Thank you.
Leslie Morgan
vi


TABLE OF CONTENTS
CHAPTER
I. INTRODUCTION..............................................................1
Background and Review of Literature.......................................1
Overview............................................................1
Innovation..........................................................1
Impact of Asthma..........................................................5
Public Health Significance..........................................5
Current Therapeutic Strategies......................................6
Pathophysiology of Asthma.................................................7
Allergic Inflammation...............................................7
Airflow Obstruction in Asthma.......................................9
Airflow Obstruction: Contractile and Non-Contractile Components....10
Smooth Muscle...............................................10
Edema.......................................................10
Mucus.......................................................10
Mucus Hypersecretion in Asthma...........................................11
Pathological Findings in Fatal Asthma..............................11
Sources of Mucus and Ontogeny of Mucin-Producing Cells.............12
Production.........................................................15
Secretion..........................................................16
Mucolytic Therapy..................................................17
Scientific Premise.......................................................21
vii


Specific Aims
21
II. MUCOLYTICS IN A MOUSE MODEL OF ALLERGIC ASTHMA...........................23
Rationale..............................................................23
Research and Design Methods............................................23
Mice.............................................................24
Asthma Model.....................................................24
Surgical Preparation.............................................26
Drug Administration..............................................27
AHR Measurements.................................................27
Histology........................................................28
Muc5b Protein Assays.............................................30
Statistical Analysis.............................................31
Results................................................................32
AHR Measurements.................................................32
Histology and Western Blots......................................42
Discussion.......................................................47
III. FUTURE DIRECTIONS.....................................................52
Microscopy Methods.....................................................52
Microscopy Results.....................................................54
Carbon Nanoparticle Methods............................................57
Conclusions and Impact of Findings.....................................60
REFERENCES....................................................................61
viii


APPENDIX
A. Protocols for flexiVent and Western Blots................................67
B. Flexivent Raw Data........................................................81
C. R Code....................................................................86
IX




CHAPTER I
INTRODUCTION
Background and Review of Literature
Overview
Asthma is characterized by airway inflammation, smooth muscle contraction, and mucus hypersecretion that collectively contribute to airflow obstruction. Airway hyperresponsiveness (AHR) is defined as an exaggerated degree of airflow obstruction in response to bronchoconstricting agents in asthma patients in comparison to non-diseased control subjects. Accordingly, compared to healthy individuals, people with asthma have a lower threshold to inhaled methacholine (MCh) (Bates et al. 2009), which is observed as potentiated airflow impairment in response to lower doses of MCh. AHR is present in asthma patients at baseline, and AHR correlates with asthma exacerbation magnitude and frequency. Innovation
Current dogma emphasizes the significance of inflammation and smooth muscle contraction as the main causes of airflow obstruction in asthma (Fanta 2009). However, postmortem studies conducted on patients who died of acute asthma, showed marked mucus airway occlusion (Fig. 1) (Evans and Koo 2009; Kuyper et al. 2003). Airflow obstruction can lead to air trapping causing lung hyperinflation (Fig. 2). These features are evident in the lungs of a 51 year old female fatal asthma victim (Fig. 3). Collectively, these pathological findings demonstrate both lung hyperinflation (the result of airflow obstruction) and mucus plugging. Mucus overproduction has been shown to occur in patients with even mild to moderate disease (Ordonez et al. 2001). Nonetheless, since mucolytics do not appear in the normal treatment regimen for asthma, their usefulness in asthma is poorly understood. In
1


Figure 1. Mucus plugging in a fatal asthma attack. Segmental bronchus in fatal adult-onset asthma, showing occlusive mucoid plugging (arrow) (Sidebotham and Roche 2003).
2


Figure 2. Lung hyperinflation in fatal asthma. Right lung from fatal asthma in a young adult. The lung remained inflated after removal with sharp margins to the lobes. The imprints of the ribs and intercostal spaces produced a corrugated effect on the lateral pleural surfaces of the lung (Sidebotham and Roche 2003).
3


Figure 3. Mucus plugging and hyperinflation in a 50 year old fatal asthma victim. Lungs from a fatal asthma patient showing regional hyperinflation in the right upper lobe (left, blue arrow) compared to the left lower lobe (left, black arrow). A mucus plug occluding a large airway (right, arrow).
4


part, this is due to an incomplete understanding of the chief macromolecular components of mucus, polymeric mucin glycoproteins.
To this end, a recent study found that the mucin protein Muc5ac was required for AHR in mouse models of asthma (MUC in humans; Muc in mice) (Evans et al. 2015). Although these studies suggest that protection in asthma could be gained by preventing mucus hypersecretion, it is not known if effective therapeutic efficacy can be achieved with mucolytics, whose benefit is gained by reversing the effects of mucus after it is secreted. Accordingly, testing mucolytic agents may determine if they will inhibit the effects of Muc5ac in the airways of mice, and thereby provide a rationale for their development as therapeutics in humans. In this vein, therapies could focus on using mucolytics to directly reduce airway occlusion by mucus, or they could be used as adjunct treatments to enhance the delivery and efficacy of mainstay drugs such as bronchodilators and glucocorticoids.
Impact of Asthma
Public Health Significance
Asthma affects over 25 million people in the United States alone, including 7 million children (CDC: Asthma Fastats 2016). According to the Centers for Disease Control, asthma poses both health and economic concerns. In 2010, asthma costed approximately $56 billion, caused 10.5 million missed days of school, and resulted in 14.2 million missed days of work in the United States (CDC: Asthma Fastats 2016). Missed school and work days can have profound impacts on entire families, especially if the family is economically disadvantaged. Furthermore, the proportion of people with asthma has increased by 15% in the past decade. In aggregate, effective treatments are needed to reduce the number of people affected by asthma and lessen the impact that it has on our society.
5


Despite the staggering cost of asthma in the United States, it is important to note that not all people with asthma do not receive adequate treatment, or in some cases any treatment at all. Black and Puerto Rican children are more likely to have asthma than white children (CDC: Asthma Fastats 2016). Asthma is also more prevalent in persons that did not attain a high school diploma, and those living below the poverty level (CDC: Asthma Fastats 2016). Unfortunately, there are dramatic differences in the care of people with asthma when considering these demographics. For instance, nearly one in four black adults cannot afford routine doctor visits and asthma medications. As a result, they are two to three times more likely to die from asthma than any other racial group (CDC: Asthma Fastats 2016). Developing new compounds that will be effective at dissolving mucus plugs will be instrumental at reducing asthma deaths, a major public health concern especially for people that fall into disadvantaged demographic groups.
Current Therapeutic Strategies
Most drugs used to treat asthma fall into one of two categories based on the following activities: (1) controlling inflammation or (2) controlling bronchospasm (Zdanowicz 2007). On the other hand, mucus is often ignored or undervalued in its role in asthma: Mucus was described as the ugly sister to bronchospasm and inflammation (Rogers 2004). In part, this is because the use of existing mucolytics has been shown to be largely ineffective (Aliyali et al. 2009), or even detrimental, with increased airflow obstruction noted via spirometry (Hirsch et al. 1967). Because the acid dissociation constant (pKa) of current mucolytics for breaking disulfide bonds is not advantageous, high concentration of drug is needed to be effective (Aliyali et al. 2010; Hays and Fahy 2003). Higher concentrations of drug would be more likely to elicit bronchospasm, which is to be avoided in people with asthma. As a result,
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mucolytics are rarely used in the treatment of asthma and treatment for mucus plugs in severe asthma is mostly supportive (Hays and Fahy 2003).
Pathophysiology of Asthma
Allergic Inflammation
Asthma can be characterized by inflammation and airflow obstruction. Inflammatory triggers include particulates, infectious agents, and allergens. Because allergic asthma is very common and is easily and reproducibly modeled in mice, my work focuses on studies in a mouse model of asthma that is driven by allergic inflammation.
Allergic inflammation consists of an early and a late phase. When a person with asthma inhales an allergen that they are sensitized to, the allergen is recognized by immunoglobulin-E (IgE) which binds to mast cells, immediately triggering mast cell degranulation (Carroll et al. 2002). Mast cells release their contents, which include inflammatory mediators such as histamine, leukotrienes, prostaglandins and plateletactivating factor (Rogers 2004). These mediators induce bronchospasm and can promote early phase inflammation.
The late phase is characterized by eosinophilia and airway remodeling that is typified by mucous cell metaplasia, a process driven by the induction and overproduction of the mucin glycoprotein MUC5AC by conducting airway surface secretory cells (Caramori et al. 2004; Evans et al. 2015; Ordonez et al 2001). Accordingly, post mortem exams of fatal status asthmaticus show pathologic features such as chronic inflammatory changes, eosinophilic infiltration, smooth muscle hypertrophy, and airway remodeling with mucus cell metaplasia (Fig. 4) (Aikawa et al. 1992; Gronenberg et al. 2002; Sidebotham and Roche 2003).
7


Figure 4. Post mortem pathological features in fatal asthma. Bronchiole in a fatal asthma patient indicating inflammation of the airway wall (green arrow), goblet cells in the epithelium {red arrow) and mucoid plugging of the lumen {black arrow), with intraluminal eosinophil leucocytes (Sidebotham and Roche 2003).
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The validity of the present studies is reliant on the accuracy of our animal model for human asthma. Allergic asthma has been studied in numerous animal models including large animals (horses and sheep) and smaller animals including dogs, rabbits, guinea-pigs, rats, and mice. Each has advantages and disadvantages. Large animals have the benefit of exhibiting spontaneous asthma but incur much higher costs to study. Smaller animals allow for studies with higher numbers, with greater control of their genetics (inbred and genetically-engineered strains), and with much lower costs to the investigator. Mice have become an obvious and convenient animal model for asthma due to the plethora of tools and techniques available to study them along with a decreased cost and relatively short gestation period.
A challenge with small animals is that they do not exhibit spontaneous asthma.
Rather, they are sensitized using a variety of triggers including chicken egg albumin, house dust mite, cockroach, plant material, and fungi. For this study, a mouse model of allergic asthma was developed by a series of challenges with aerosolized fungal allergen made from an Aspergillus oryzae extract (AOE). Once the challenges were complete, mice were studied during the late phase of inflammation 48 hours after the last AOE challenge.
Airflow Obstruction in Asthma
In humans, reductions in airflow can be measured by a forced expiratory volume over one second (FEVi) of less than 80% of predicted normal based on the persons sex, age, and height. In acute asthma attacks, airflow obstruction can be reversible to some degree, as evidenced by an increase in FEVi of greater than 12% following inhalation of a P2 adrenergic receptor agonist (Egan et al. 2009). Decreased expiratory flow rates are the result of airway lumen narrowing and occlusion in the conducting airways. Patients with asthma have been noted to have AHR, which is defined as increased sensitivity of the airways to
9


bronchoconstricting agents. This narrowing can be due to smooth muscle-mediated bronchoconstriction, or to non-contractile obstructions, such as airway edema, inflammation, and excessive mucus in the airway, or to a combination of both contractile and non-contractile factors.
Airflow Obstruction in Asthma: Contractile and Non-Contractile Components
Smooth Muscle
It is clear that the heightened sensitivity of asthmatic patients to MCh reflects contractions of the airway smooth muscle. Once a patient has established airway inflammation and AHR with asthma, smooth muscle-mediated bronchoconstriction can be triggered by a variety of endogenous factors as well. These include, but are not limited to: inhalation of irritants or cold air, exercise, emotional stress, hyperventilation, and pharmacologic agents such as methacholine and histamine (Egan et al. 2010). In addition to this well-recognized contractile component, non-contractile factors also result in airflow obstructions.
Edema
Non-contractile airflow obstruction can be caused by edema related to inflammation or by excessive mucus secreted into the airways. Inflammation resulting from infection or allergen exposure can result in swelling of the tissues that surround bronchial airways, causing the airway walls to expand, thereby narrowing the passage of air. Edema may be sensitive to glucocorticoid treatments, which are anti-inflammatory and presumably reduce swelling.
Mucus
The other prominent, non-contractile feature of airflow obstruction in people with asthma is excessive mucus in the airways. In non-diseased states, mucus is protective and
10


necessary for airway defense (Roy et al. 2014). The mucus layer coats the airway over a carpet of cilia that propel mucus and materials contained within mucus to the oropharynx, where harmful foreign particles can be eliminated by expectoration or swallowing. This system is known as the mucociliary escalator (Egan et al. 2009; Knowles and Boucher 2002), and it appears to exist as a periciliary gel-on-brush structure (Button 2012). Changing the abundance or the viscosity of mucus can adversely affect these protective mechanisms (Fahy and Dickey 2011; Sheehan et al. 1995; Yuan et al. 2015).
Mucus Hypersecretion in Asthma Pathological Findings in Fatal Asthma
In individuals with chronic lung disease, the normal mechanisms of airway defense are disrupted, in part through overproduction of mucins. As a result of the increase in mucin production and secretion, the airway accumulates more mucus, which can diminish or occlude the airway lumen (James & Carroll 1995; James et al. 1989). When combined with the effects of smooth muscle-mediated bronchoconstriction, this occlusion can dramatically potentiate airflow obstruction, or be fatal (Carroll et al. 2002; Sheehan et al. 1995). In autopsied lung tissues from patients who die of fatal asthma episodes, airway occlusion by mucus is noted to be a ubiquitous feature (see Figs. 1 and 2) (Gronenberg et al. 2002; Kuyper et al. 2003; Sheehan et al. 1995; Sidebotham and Roche 2003). Consequently, there is a need for pharmacologic compounds that can control mucus in asthma and potentially treat patients once they have progressed to a state of mucus hypersecretion with mucus plugging.
In fatal asthma attacks, the mucus of the victims was found to be abnormally solid in its composition (Sheehan et al. 1995). Studies conducted as early as the 1920s, when many diagnostic tools were not available, were able to make visual observations of this
11


phenomenon post mortem (Houston et al. 1953; and Huber and Koessler 1922; James et al. 1989). With mucus plugging comes hyperinflation and air trapping as shown in Fig. 4. According to Sidebotham and Roche (2003), asthma is a preventable cause of mortality caused by a knowledge gap that persists despite our advanced understanding of the disease, and the pharmaceutics available for treatment, (Sidebotham and Roche 2003). These authors attributed these deaths to patients being misdiagnosed and undertreated (Sidebotham and Roche 2003).
Indeed, despite the fact that almost all post mortem studies of fatal asthma described mucus airway occlusion as the cause of death, there are currently no efficacious mucolytics on the market. In order to research potential mucolytic compounds, we need to investigate these mucin proteins more closely at molecular and biochemical levels. Recent studies show that mucus secretions in fatal asthma are unusually tenacious, due to several different factors including the high concentrations of mucin proteins, their low charge density, and their extremely large size, their (Sheehan et al. 1995).
Sources of Mucus and Ontogeny of Mucin-Producing Cells
During healthy airway conditions, mucus is comprised of mostly water (~ 98%) and is defined as, the free slime of mucous membranes, composed of glandular and surface cell secretions, along with various water, inorganic salts, desquamated cells and leukocytes, DNA, and polypeptides (Bansil and Turner 2002; Evans & Koo 2008; Sheehan et al. 1995). There are several types of mucin secretory cells in the human airway. Of particular note are the goblet and club cells (formerly known as Clara cells). Goblet cells are more abundant in the upper airways, and virtually absent in the lower airways (Boers et al. 1999; Rogers 2003) Club cells are scant in the upper airways, and more prominent in the lower airways (Boers et
12


al. 1999). Both of these mucin-secreting cells have been shown to increase in number and size in states of chronic inflammation (Boers et al. 1999; Carroll et al. 2002; Dunnill et al. 1969; Evans et al. 2004, 2009; Rogers 2003). These increases can be problematic to the normal protective and clearance mechanisms of the human airway (Button et al. 2012).
It has been shown that the number of club cells present in health is as follows: the terminal bronchioles, airway generation 15, contain about 11% club cells, and the respiratory bronchioles, airway generation 16, contain about 22% club cells (Boers et al. 1999). In allergic disease states such as asthma, there can be a dramatic increase in the amount of mucin-producing cells in humans as well as mice (Evans et al. 2004; Ordonez et al. 2001). This increase results from allergic airway inflammation causing the production and storage of mucins, particularly MUC5AC/Muc5ac (Evans et al. 2004; Ordonez et al. 2001).
In aggregate, the increase in mucin production by secretory cells throughout the conducing airways results in increased numbers of mucin-producing goblet cells, a phenotype that is referred to as to mucus cell metaplasia or hyperplasia. Patients with asthma have increased of mucin secretory cells compared to healthy individuals (Fig. 5). These changes can be very dramatic. Aikawa et al. (1992) show a 30 fold increase of goblet cells to total epithelial layer in three patients who had died of status asthmaticus. This same study also found that the cilia in these victims airways were decreased in both number and length. These findings could contribute to the diminished capacity of the mucociliary escalator, leading to an increased amount of retained mucus (Aikawa et al. 1992). Importantly, mucus hypersecretion also appears to be prominent in mild-to-moderate disease. Ultimately, these factors lead to the release of excessive amounts of mucin glycoproteins into the airways in response to several triggers in asthma (Thornton and Sheehan 2004).
13


Figure 5. Mucin secretory cell metaplasia. Photomicrographs of representative histologic sections from endobronchial biopsies from a healthy subject (left) and a subject with asthma (right). Mucin stores in goblet cells appear as a mixture of blue and purple staining. The sections demonstrate increased Alcian blue and PAS staining in the section from the subject with asthma (Ordonez et al. 2001).
14


Production
Mucin glycoproteins are the predominant macromolecules in mucus and are responsible for its viscoelastic gel-like properties (Bansil and Turner 2002). There are roughly nineteen mucin proteins encoded in the human genome. The majority of these are cell tethered mucins; the rest are secreted mucins (Thornton et al. 2008). Among the secreted forms, four polymeric/gel-forming mucins have been shown to be expressed in the human airway-MUC2, MUC5AC, MUC5B, and MUC19 (Evans & Koo 2009). Based on their expression levels and functional significance in humans and mice, the two polymeric mucins that play the most prominent roles are MUC5B/Muc5b and MUC5AC/Muc5ac.
In mice, Muc5b is essential for the formation of a protective mucus gel (Roy et al. 2014), demonstrating that it is required for homeostatic defense. By contrast, Muc5ac is dispensable for baseline airway defense. However, after allergen challenge, the airways can produce excessive levels of MUC5AC/Muc5ac (Evans et al. 2004; Thornton et al. 2008). In human airway epithelia, MUC5AC increases in the presence of chronic inflammation and is induced in asthma (Gronenberg et al. 2002; Ordonez et al. 2001). Further, Muc5ac was shown to be required for AHR in mouse models of asthma. In Muc5ac knockout (Muc5ac "") mice that had been challenged with aerosolized AOE for four consecutive weeks, the airways presented with marked inflammation, but failed to have significant AHR in response to inhaled MCh (Evans et al. 2015). The data above suggest that MUC5AC would be an appropriate target for future pharmacologic compounds.
Inhaled corticosteroids have traditionally been used to combat airway inflammation, and they have also been shown to reduce MUC5AC expression and mucous metaplasia. The anti-mucous mechanism of action of corticosteroids is to modify the transcription of
15


inflammatory mediators, thereby reducing inflammation, and potentially to also directly repress MUC5AC promoter activity and gene expression (Zdanowicz 2007). Indeed, glucocorticoid treatment has been shown to potently reduce MUC5AC gene expression in human lung epithelial cells in vitro (Chen et al. 2006, 2012, 2014; Takami et al. 2012;
Hauber et al. 2007; Lu et al. 2005). However, in vivo studies of Muc5ac expression and mucous cell metaplasia in animal models have resulted in mixed findings with steroid treatments. Some studies show strong suppression of inflammation (Blyth et al. 1998; Lundgren et al. 2009) and others show mild (Yamabayashi et al. 2012; Bos et al. 2007) or no suppression (Mushaben et al. 2013; Kibe et al. 2003). It is not clear whether these conflicting results are due to species differences or the nature of experimental challenges. Thus, while the anti-inflammatory effects of corticosteroids are well-established, their effects on mucin expression are variable and in many cases not efficacious once mucus secretion is already up-regulated. Hence, at the very least these results reduce support for the utility of glucocorticoids as an effective strategy to reduce MUC5AC production and mucous cell metaplasia in asthma a direct anti-mucus intervention is needed.
Protein Assembly and Secretion
Mucin production describes the manufacturing process of the mucin proteins in secretory cells. Secretion, on the other hand, describes the release of mucin proteins on to the airway surface. There are several triggers for acute secretion such as, inflammatory mediators and neurotransmitters (Evans and Koo 2008). These triggers are activated by the AOE challenges, and administration of MCh. While mechanisms of secretion are being studied by other groups (Adler et al. 2013), in this study, I investigated the efficacy of a mucolytic that acutely blocks the effects of a post-secretory event.
16


Secreted mucin proteins are large (2-50 megadaltons), complex, highly glycosylated, protein polymers (Innes et al. 2009). A large portion of their mass is carbohydrates and these glycan chains form the large central domain of the mucin protein that absorbs copious amounts of water, leading to mucus with strongly viscous properties (Fig. 6) (Thornton et al. 2008). Polymeric mucins, including MUC5AC/Muc5ac and MUC5B/Muc5b, have cysteine rich domains at their N- and C- termini, which are responsible for disulfide mediated gel forming properties (Thornton et al. 2008). These disulfide bonded termini can form long chains and branches of mucin oligomers (10s to 100s of microns long). Intramolecular disulfide bonds can also form in centrally located the cysteine rich domains. These bonds lead to the development of extremely high molecular weight polymers that have elastic properties. Under healthy conditions the amounts of polymeric mucins and water are in equilibrium, creating a protective gel that is viscoelastic. However, the presence of excessive mucin solids causes mucus to thicken dramatically, and can result in the formation of tenacious mucus plugs that are transported poorly.
Mucolytic Therapy
Mucolytic Therapy Aerosolized mucolytics could target the apoprotein backbones, glycans, or disulfide bonds. An ideal mucolytic would thin mucus, making it is easier to expectorate. This would, in effect, aid the normal defense mechanisms of the airway and reduce mucus plugging. The use of a mucolytic could clear unwanted secretions from the airways, which would increase airflow. This, in turn, could potentiate the effects and benefits of beta agonists and corticosteroids.
A protease-based mucolytic could be a useful pharmaceutical agent to disrupt the mucin polymer backbone. Research by Innes et al. (2009) showed that the thick mucus
17


nmm mm n 1 n n


rm 1 n 11 | 1 1 1

Figure 6. Polymeric Mucin Proteins MUC5AC and MUC5B. The apoprotien backbones of MUC5AC and MUC5B are comprised by an NH2 termini (light grey) and COOH termini (dark gray). The central portions are comprised of a mucin glycosylation domain (green). This region is rich in serines and threonines that are sites of 0-1 inked glycan attachments.
The glycosylation domain is interrupted by additional cysteine-rich CysD domains (red). A: The COOH-terminal von Willebrand-like domain (VWD) is the site of disulfide dimerization. Dimers can also assemble at the NH2 termini via covalent disulfide bonds. These dimers are ~1 pm in length. B: Intramolecular disulfide bonds can also assemble in the cysteine-rich CysDs domains. This occurs when hydrophobic amino acids form loops that may promote a mucin mesh network. C: In the mucin glycosylation domain, glycan linkages occur on the hydroxylated ends of serine and threonine residues through the initial attachment of N-acetylgalactosamine (GalNac). This initial O-glycosylation step is followed by galactose and/or N-acetylglucosamine (GlcNac) attachments, and can be elaborated further by the attachment of fucose, sialic acid, and sulfate end groups (Evans et al. 2016).
18


present in acute asthma is laden with plasma proteins, which inhibit the degradation of mucins in a protease-dependent manner (Innes et al. 2009). The polypeptide extensions that emerge from these domains are instrumental to the development of the mucin gel network, which leads to increased viscosity (Thornton et al. 2008).
Another consideration might be to use a glycan inhibitor that would target the numerous glycosylated mucin domains. Current research into the glycans on mucin proteins is of current interest. There is some evidence for selective glycosylation of airway mucins in asthma, but these are poorly characterized and not adequately targeted.
Disrupting mucus polymers by targeting disulfide bonds could loosen dense mucus aggregates and thereby restore healthy mucus functions (Fig. 7). Indeed, at present, the best-characterized, potentially safest, and most efficacious options are agents that work by breaking up the disulfide bonds of mucins (reducing agents). One such mucolytic currently on the market in the United States is N-acetylcysteine (NAC, brand name Mucomyst). NAC works by breaking up the disulfide bonds of mucus into weaker sulfhydryl bonds (Egan et al. 2010; Gardenhire and Rau 2008). This decreases the viscosity of the mucus, so it can be expectorated. N-acetylcysteine has a short half-life on airway surfaces, and it is a weak reducing agent at physiologic pH. For any therapeutic benefit to be seen, NAC must be given as an aerosol at a 1 M concentration, which has the adverse effect of irritating airways and triggering bronchospasm.
Because of its potential as an effective therapeutic agent, our collaborators at Parion Sciences, as well as colleagues in other academic laboratories (Yuan et al. 2015), have placed effort into developing mucolytic agents that disrupt disulfide bonds with greater potency. My research plan seeks to determine whether delivering the phosphine compound P-3001, a fast-
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Pathologic mucus
Pathologic mucus + P-3001
Healthy mucus
# = Mucolytics dissolving disulfide bonds
Figure 7. Mucolytic action on disulfide bonds. Disulfide bonds shown as both end to end
formations (s-s) at the N and C termini, and as intramolecular formations in the cysteine rich domains (s-s), contributing to thick and tenacious mucus (left). Application of P-3001, a phosphine based reducing agent, could target and dissolve these bonds (middle), making mucus less viscous and easier to expectorate (right) (modified from Yuan et al. 2015).
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acting mucolytic that is at least 500-fold more potent than NAC, disrupts the mucin polymer network, thus suggesting that inhaled mucolytic therapy is an effective potential pharmacologic approach in the treatment of asthma and potentially other obstructive diseases.
Scientific Premise
Asthma is a serious and preventable disease. Despite our advanced understanding of the pathophysiology of asthma, and the pharmaceutics available for its treatment, its prevalence remains high, and patients still die from this disease (Sidebotham and Roche 2003). Despite the fact that almost all post-mortem studies of fatal asthma described mucus airway occlusion as the cause of death (see Fig. 1), mucolytics are not being added to asthma treatment regimens. This is due in part to the limited availability of agents that are efficacious at tolerable concentrations (Gronenberg et al. 2002). This study seeks to determine whether a mucolytic agent with a similar mechanism of action to NAC, but with greater potency at breaking disulfide bonds, could provide support for the efficacy of disrupting mucin polymer to improve airflow in an allergic asthma model.
Specific Aims
The goal of this study was to create a mouse model of asthma that would be useful for determining whether a pharmacologic agent is effective in treating mucus secretion, and to test the effects of mucolytic treatment on AHR.
Aim 1. Create an allergic mouse model with mucus dependent AHR that will permit testing of the effects of mucolytic intervention. I adapted an existing protocol for studying AHR in AOE challenged WT mice. This allowed the simultaneous testing of a bronchoprovocative agent, mucin secretagogue, and mucolytic intervention treatments.
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Aim 2. Determine the effects of mucolytic intervention on AHR. Employing the model established above Aim 1, the effect of an inhaled reducing gent P-3001 on AHR was tested.
Aim 3. Determine the effects of mucolytic treatment on airway mucus plugging and mucin polymer structure. Histologic data was used to quantify the extent of inflammation and mucus hypersecretion present in the mouse models. Western blots were done to determine the extent of mucin protein breakdown in bronchoalveolar lavage samples post-delivery of P-3001 or saline.
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CHAPTER II
MUCOLYTICS IN A MOUSE MODEL OF ALLERGIC ASTHMA
Rationale
Mucus is a defining feature of asthma in humans (Gronenberg et al. 2002); and mucus plugging is a characteristic of fatal asthma attacks (Kuyper et al. 2003). In mice, Muc5ac is essential to allergic asthma-like obstruction since Muc5ac_/" mice have previously been shown to be protected against AHR to inhaled MCh (Evans et al. 2015). The long-term goal for my research is to determine whether an inhaled mucolytic could ultimately be beneficial for patients with asthma.
This study is the first to deploy a mucolytic intervention in a mouse model of allergic asthma. The experiments presented here were designed to establish a suitable model (Aim 1), demonstrate the efficacy of a test compound (Aim 2), and verify that the compound affected its target (Aim 3). Accordingly, the studies below test the central hypothesis that a fast acting mucolytic agent will prevent AHR by acutely disrupting the formation of mucus plugs.
A mouse model of allergic asthma was developed by a series of challenges with aerosolized AOE. Once the challenges were complete, mice received increasing dosages of intravenous MCh in the presence of aerosol administration of either saline or the mucolytic P-3001. Lung mechanics were measured in mechanically ventilated mice. Total respiratory system resistance (Rrs) was calculated, and differences in Rrs were determined between allergically-inflamed and MCh-treated mice that received P-3001 and those that did not. In addition, histology and Western blots were done to quantify the amount of mucus present in the airways, and to demonstrate the breakdown of mucin proteins among the two test groups.
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Research and Design Methods
Mice
All studies were conducted with the approval of the University of Colorado Denver Institutional Animal Care and Use Committee (IACUC). For studies in wild type (WT) animals, BALB/cJ mice were used, as this strain is well-studied in modeling allergic asthma. In addition, Muc5ac _/" mice that were crossed onto a congenic BALB/cJ strain background were used to generate data for comparison to previous studies (Evans et al. 2015). Both male and female mice were used beginning at 6-8 weeks of age. Mice were housed in specific pathogen-free conditions in ventilated cages with no more than five mice per cage. Mice were kept on a 12-hour light and dark cycle with the light cycle being 0600-1800. All mice were fed irradiated normal mouse chow and had access to food and water ad libitum. Asthma Model
To model asthma-like inflammation and mucous cell metaplasia, mice were challenged using aerosolized Aspergillus oryzae extract (AOE; Sigma, catalog # P6110). AOE is a protease complex produced by submerged fermentation of A. oryzae. AOE has been shown previously to elicit an allergic inflammation in mice (Kheradmand et al. 2002; Evans et al. 2015). Aerosolized AOE challenges were given using an Ultravent jet nebulizer which generates a particle size of <1 pm. Mice were loaded into custom built nose-only exposure chambers, and a challenge aerosol was generated using 5 ml of a 10% vol/vol mixture of AOE and saline. Challenges lasted until the full volume was delivered (approximately 40-45 minutes) (Evans et al. 2015). Mice received 4 weekly challenges, and endpoint analyses were studied 48 h after the last AOE challenge, which corresponds with a late phase asthmatic response (Fig. 8). Mouse challenges provide our model with inflammation, mucous cell metaplasia with associated mucus hypersecretion, and AHR.
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Base unit Module flexiWare software
Figure 8. Immunization and Aerosol Challenge protocol for Inducing Allergic Phenotypes and the flexiVent. BALB/cJ mice were challenged weekly with AOE. Measurements were then determined 48 hours after the last challenge (top). The FlexiVent small animal ventilator (bottom) (Scireq, Montreal Quebec, Canada).
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Surgical Preparation
The techniques and measurements in my model of asthma present a challenge due to the small size of the animal. Whereas human asthma is easily tested using non-invasive procedures, noninvasive methods of attaining lung mechanics data in mice are not as accurate as invasive ones. Invasiveness does dictate that mice are studied under conditions that are far from a natural state (Bates & Irvin 2003), but the dichotomy between measurement precision and maintaining natural conditions was thoughtfully considered in the design of these studies. In this instance, experimental control and measurement precision was chosen over noninvasiveness. Assessments were made with the flexiVent (Scireq, Montreal Quebec, Canada), a ventilator used for measuring changes in respiratory system resistance (Rrs) and responses to inhaled and intravenous (IV) drugs (see Fig. 8). These experiments were conducted in anesthetized, tracheostomized, paralyzed mice using the single-frequency forced oscillation technique (SFOT), in which a sinusoidal oscillatory flow signal is applied to the airway opening while the airway pressure is measured (Bates et al. 2009).
Mice were anesthetized with a 2 g/kg (0.01 ml/g) intraperitoneal (IP) dose of urethane. Urethane provides deep sedation that has been shown previously to have a low impact on cardiac and respiratory systems (Soma 1983). Furthermore, urethane induced anesthesia lasts for up to 2 hours, although each experiment lasted approximately 20 minutes (Hara and Harris 2002). Once anesthetized, surgery was performed to cannulate the trachea with a beveled 18 gauge blunt tip catheter. Mice were then placed on the flexiVent. Ventilated mice were paralyzed with an initial IP injection of 0.2 ml of a 20 mg/ml solution of succinylcholine chloride to prevent spontaneous respirations from interfering with Rrs
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measurements. In order to maintain paralysis throughout the experiment, the initial bolus was followed by a continuous infusion of succinylcholine administered IV.
Drug Administration
Once mice were paralyzed and stably ventilated, their abdomens were surgically opened for catheterization of the inferior vena cava (IVC), followed by a continuous infusion of succinylcholine chloride (50 pg/ml solution). Continuous IVC infusion also contained a 5 pl/g dose of methacholine (MCh) diluted in phosphate buffered saline (10 pg/mL solution) and succinylcholine chloride (20 pg/ml). MCh is a synthetic cholinergic that stimulates muscarinic receptors to cause smooth muscle contraction and mucin secretion. MCh challenges for AHR studies can be administered as an aerosol, or by IV routes. In order to rule out potential competition between MCh and mucolytic agents if they were aerosolized simultaneously, administration routes have been separated, and MCh was delivered IV while mucolytic treatment was delivered by aerosol. Mice were then given either aerosolized sterile normal saline, or aerosolized mucolytic diluted in saline. Aerosols on the flexiVent were administered with an in-line ultrasonic nebulizer (see below).
AHR Measurements
After placement on the flexiVent, 2-3 deep inflation maneuvers were performed to facilitate alveolar recruitment and normalize lung volumes (Fig. 9) (Hantos et al 2003). Baseline values of respiratory mechanics were then assessed with several perturbations using 2.5 Hz single frequency forced oscillation techniques (SFOT). Each cycle included a default ventilation pattern consisting of: respiratory rate = 150/min, tidal volume = 10 ml/kg, pressure limit = 30 cird/hO, PEEP = 3 cird/hO, Inspiratory-time = 0.16 s, and Expiratory-time = 0.2 s. In addition to the default ventilation pattern that ran the length of the experiment,
27


several perturbations were executed per an automation script. These included, in order, two deep inflations, administration of aerosolized medication, and a Snapshot-150 perturbation to determine Rrs for each of the 6 doses of MCh. Rrs is calculated in the flexiVent software by fitting the equation of motion of the linear single compartment model of lung mechanics to a SFOT data using multiple linear regressions (Shalaby et al. 2010).
Since asthma is characterized by abnormal resistance to airflow in the lungs, as well as exaggerated responsiveness to agents that acutely cause obstruction, changes in Rrs were measured in response to increasing doses of MCh (Mcgovern et al. 2013). AHR was defined as exaggerated Rrs responses to given doses of MCh between animals. To determine this, differences in Rrs changes in response to MCh were analyzed by performing linear regression on log-transformed dose-response curves and testing for differences in the slopes of the regression lines for each mouse in each of the treatment groups.
After the last dose of MCh was delivered, mice were removed from the ventilator and euthanized by exsanguination. Mice were subsequently subjected to fixation of the lungs with a methanol-based fixative consisting of: 60% methanol (100%), 30% chloroform, and 10% glacial acetic acid, for histology, or a bronchoalveolar lavage (BAL) for Western blots. Histology
Fixed lungs were excised after 30-60 minutes and placed in a scintillation vial filled with methacam. After 24 hours, lungs were removed and placed in a scintillation vial with 100% methanol. Methanol solutions were replaced with fresh solution again in 24 hours. After fixation in methanol, left lungs were cut into ~2 mm transverse sections and imaged. Lung volume was calculated using the Cavalieri method, or Archimedes principle (Krefft et al. 2015), followed by embedding in paraffin, and sectioning into 5 pm thick specimens that
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Mr
(MCta, 4, 8,16,32,64,128, gg'kg, Iv)
100mMP3001via aerosol between closes
~5 minutes between P3001 closes
Estimate delivers
~1.5 gl per aerosol (6 nmol per gram)
Figure 9. FlexiVent script perturbations with dosages of MCh and P-3001. Mice received a default ventilation pattern of 150 breaths/min. After placement on the flexivent, and prior to aerosol delivery, two deep inflations were administered (top left). A snap shot measurement, denoted by the sinusoidal waveform (top right), from which respiratory system resistance (Rrs) is derived. This cycle of Rrs measurements is repeated 20 times for each dose of methacholine (MCh). Dosages of MCh and P-3001 shown respectively (bottom).
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were collected on positively charged glass microscope slides. Tissues were stained with alcian blue periodic acid-Schiff s (AB-PAS) stain and examined with an Olympus BX63 microscope in order to establish degrees of mucin production and secretion. This was done to determine if mice treated with the mucolytic P-3001 showed more patent airways, compared to mice treated with only aerosolized saline.
Muc5b Protein Assays
Western blots were performed on BAL specimens to determine the changes in mucin polymer sizes of specimens from mice that received P-3001 versus those that received only aerosolized saline. This was done to determine if P-3001 was effective at breaking down the mucin glycoproteins in the bronchoalveolar fluid (BALF) obtained after the flexivent experiments. BALF samples were first thawed in an ice/water slurry. A protease inhibitor was added to each sample (10% per sample volume). BALF for each sample and double deionized water were added to 1.5 ml tubes in order to normalize for concentration or pg of protein per lane, based on analysis from the dot blot, for a total of 25 pi per tube. 5 pi of loading buffer was added to each tube, and samples were loaded into the wells of a prepared agarose gel. The loaded agarose gel was covered with a running buffer, consisting of lx Tris-Acetate + ethylenediaminetetraacetic acid (EDTA) (TAE) + 0.1% sodium dodecyl sulfate (SDS), and allowed to run overnight in a 4 C cooler at 42 V.
On day two, the agarose gel was placed in a transfer apparatus with a PVDF membrane and covered with saline sodium citrate (SSC). The apparatus was attached to a vacuum pressure of -25 to -37.5 cmFbO for 90 minutes. Membrane was then removed and rinsed 2-3 times in 4x SSC and blocked with Odyssey buffer for one hour on a rocker.
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Primary antibody (rabbit-anti-mouse Muc5b; 1:5000 dilution) was applied to membrane and container incubated overnight in 4C refrigerator with gentle rocking.
On day three, the primary antibody was discarded and membrane was washed 3-4 times for 5 minutes each wash with lx tris-buffered saline + 0.05% Tween 20 (TBS-T). Membrane was then probed with a secondary antibody (IRDye 680 RD Goat-anti-Rabbit; Lot# C41029-03; stored undiluted at 4C; used at 1:15,000 dilution) for one hour with gentle rocking. Membranes were protected from light with a foil covered receptacle during this phase. Washings with TBS-T were repeated as above and immunoreactivity was imaged in an Odyssey FC system (LI-COR Lincoln, NE).
Statistical Analysis
Statistical analysis and graphs were performed in GraphPad Prism 7 (San Diego, CA) and R statistical software. Data from dose response curves were log transformed, extreme outliers were removed, and linear regression was performed. Data from mice that met the exclusion criteria below were also removed. The slopes for each group were compared using a one-way ANOVA with Dunnetts multiple comparison tests.
There were a few cases in which data were excluded. Criteria for exclusion of AHR data in mice consisted of (1) death of the animal prior to the test (n = 5), (2) abnormally high baseline values not responsive to basic manipulation, such as repositioning of the animal, the platform, or the cannula (n = 3), (3) abnormally low baseline values, denoting a leak in the system with either the equipment or the animal, or incomplete paralysis resulting in spontaneous respiratory effort, that could not be readily identified and corrected with either manipulation or bolus of paralytic agent (n = 2), and (4) values that decreased and then increased again with the dose response curve (complete exclusion n = 11). For this last case,
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mice were not always completely excluded from the study. Rather, their Rrs values that increased in a dose dependent manner were included, and all subsequent values ignored under the assumption that the cause of the decrease in values was due to death of the animal. This was observed in allergically inflamed mice, and the loss of dose-responsiveness occurred after responses that were strong enough to kill the animal (evidenced by severe bradycardia that does not recover).
Results
AHR Measurements
In Aim 1,1 sought to determine whether IV MCh challenge would be useful in my study design. In response to IV MCh, AOE challenged WT mice showed higher Rrs values compared to unchallenged mice. The dose response curves showed a mean difference of approximately 8-fold in Rrs values for the AOE challenged mice compared to the unchallenged, non-allergic healthy control mice (Fig. 10). Thus, AOE mice had a potentiated increases in resistance compared to that of the unchallenged WT mouse. This confirms that the AOE challenged WT mice exhibited increased AHR.
Previous studies have suggested that IV infusion of MCh induces solely airway smooth muscle constriction, as opposed to what is observed with aerosolized administration, which can trigger smooth muscle constriction and epithelial mucin secretion in the airways (Petak et al. 1997). I thus next determined the role of mucin secretion in AHR as a response to IV MCh. It has been shown that the mucin protein Muc5ac is required for inhaled MCh induced AHR in AOE challenged WT mice (Evans et al. 2015). Therefore, I tested genetically deficient Muc5ac (Muc5ac "") mice to examine whether mucin dependent AHR to IV MCh was also present.
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Respiratory System Resistance (cmH20 ml"1 s1)
AOE
Unchallenged
4 8 16 32 64 128
MCh (pg/kg/min, iv)
Figure 10. Administration of AOE creates a suitable model of allergic asthma and AHR in mice. Airway resistance results in BALB/cJ WT mice showing an approximate eight-fold increase in respiratory system resistance (Rrs) of the AOE challenged WT mice compared to that of the unchallenged mice after AOE challenges.
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I showed that Muc5ac _/" mice demonstrated lower changes in Rrs in response to MCh than that of AOE challenged, untreated WT mice in response to IV administration of methacholine (Fig. 11). The AOE challenged, non-mucolytic treated WT mice showed higher changes in Rrs compared to AOE challenged Muc5ac _/" mice. Rrs of the AOE challenged WT mice is approximately three-fold higher than that of the AOE challenged BALB Muc5ac mice. This supports the previous finding that Muc5ac is required for AHR. This difference between the dose response curves of the AOE challenged, non-mucolytic treated WT mice and the AOE challenged Muc5ac _/" mice represents the mucus dependent component of AHR. The difference between the dose response curves of the AOE challenged Muc5ac _/" mice, and the unchallenged mice, represents the non-mucus dependent component of AHR. The most likely contributors to the non-mucus dependent component being smooth muscle-mediated bronchoconstriction and inflammation.
In aggregate, these studies in AOE challenged, Muc5ac _/" mice represent genetic prevention of AHR in response to IV MCh. Importantly, these also demonstrates that the IV MCh challenge protocol is well-suited for analysis of topical treatment with a mucolytic agent, in that I was able to elicit the mucus dependent component of AHR with IV MCh. Having established the applicability of this model, I next sought to test the effects of mucolytic treatment in acute reversal of AHR.
AOE challenged WT mice treated with aerosolized P-3001 showed substantially lower changes in Rrs in response to IV MCh compared to their AOE challenged, non-mucolytic treated littermates, and almost identical dose response curves to that the of AOE challenged Muc5ac _/" mice (Fig. 12). The Rrs values of my mucolytic treated and unchallenged groups were similar, suggesting that there was a treatment effect in the P-3001
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MCh (ng/kg/min, iv)
Figure 11. Muc5ac is required for AHR in an AOE induced allergic mouse model of asthma. Airway resistance of BALB Muc5ac "mice in response to IV MCh challenges. Airway resistance of challenged WT mice is approximately three-fold higher than that of the knockout mouse. Unchallenged and BALB WT AOE data are the same as shown in Fig. 10. The difference between the dose response curves of the AOE challenged Muc5ac KO mice, and the AOE challenged WT mice, represents the mucus dependent component of AHR. The difference between the unchallenged mice and the Muc5ac KO mice illustrates the nonmucus dependent component of AHR.
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25

20
15
10
5
0
AOE
AOE + P-3001
Muc5ac KO Unchallenged
4 8 16 32 64 128
MCh (ng/kg/min, iv)
Figure 12. P-3001 protects against AOE induced AHR in my allergic model of asthma,
Airway resistance of AOE challenged-mucolytic treated WT mice in response to IV MCh challenges. Airway resistance of AOE challenged-untreated mice is approximately three times greater than that of the AOE challenged P-3001 treated mice. Unchallenged, BALB WT AOE, and BALB MucSac data are the same as shown in Fig. 10 and 11.
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treated group. AOE challenged untreated mice had Rrs measurements that were approximately two and a half times greater than that of the AOE challenged P-3001 treated WT mice demonstrating that AHR was effectively reduced with the application of the mucolytic P-3001.
The Rrs data was log (x) transformed, outliers were removed, and linear regression was performed on the individual mice. The means of the slopes for each group in response to IV MCh are shown (Fig. 13). The mean slope of the AOE challenged, non-mucolytic treated WT mice was higher than that of the unchallenged mice 13.1 2.8 versus 1.3 0.3 respectively (data are shown SEM). The mean slopes of the AOE challenged Muc5ac mice (demonstrating genetic prevention of AHR), and the AOE challenged P-3001 treated group (demonstrating acute reversal of AHR) were 3.4 0.6 and 2.9 0.9 respectively. They were both substantially lower that the AOE challenged, non-mucolytic treated WT mice.
Individual regression slopes for each mouse after linear regression of the log (x) transformed data is shown separated by group (Fig. 14). A list of data from all mice used in this study with their respective individual slopes and r2 values (Table 1). A one-way ANOVA was performed on the individual slope values from table 1 and compared with Dunnetts multiple comparison test (Fig. 15). The figures are shown with mean SEM bars. The AOE challenged group was found to be statistically higher than all other groups of mice in this study (p-value < 0.05).
In summary, there were significant differences (p value < 0.05) in AHR between the AOE challenged P-3001 treated WT mice, versus the AOE challenged, non-mucolytic treated WT mice, with significant p-value illustrated by an asterisk (see figure 15). The AOE challenged, P-3001 treated WT mice had almost identical slopes when compared to the AOE
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Linear reg. of log(x) transformed data by group
Unchallenged
AOE
AOE + P-3001 Muc5ac KO AOE
Figure 13. Summary of AHR in allergic mice. Above are the slopes after log(x) transformation by group.
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Linear reg. of log (x)-Unchallenged Linear reg. of log (x)-AOE
Linear reg. of log (x)- Muc5ac KO AOE Linear reg. of log (x)-AOE + P-3001
Figure 14. Individual regression slopes of log (x) transformed data by group. Individual slopes for unchallenged mice shown in green (n = 7, top left). Individual slopes for AOE challenged, non-mucolytic treated WT mice shown in magenta (n = 12, top right). Slopes for AOE challenged Muc5ac KO mice shown in orange (n = 4, bottom left). Slopes for the AOE challenged P-3001 treated WT mice shown in blue (n = 7, bottom right).
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Table 1. Individual slopes and R2 values per group.
Unchallenged Slope R squared
505 2.8 0.81
506 0.7 0.93
509 1.5 0.92
516 0.2 0.99
522 -0.1 0.15
529 1.7 0.97
530 2.2 0.93
AOE
514 29.2 0.70
549 6.9 1
552 26.2 0.87
554 4.1 0.98
555 16.6 0.87
584 4.5 0.92
586 4.4 0.66
597 10.8 0.95
606 16.1 0.95
607 22.0 0.97
613 6.9 0.65
617 9.9 0.90
AOE + P-3001
556 0.1 0.29
585 1.3 1.00
598 4.6 0.96
614 -0.0 0.41
615 7.8 0.96
616 5.9 0.81
625 4.4 0.78
Muc5ac KO AOE
555 0.0 0.86
556 2.3 0.86
558 4.0 0.71
560 4.4 0.72
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Individual R rs regression slopes log (x) data
Figure 15. AOE challenged, non-mucolytic treated WT mice were significantly different than all other groups. One way ANOVA with Dunnetts multiple comparison test performed on individual slopes for each group showed that all groups were significantly (p-value < 0.05, denoted by *) different from the allergically challenged, non-mucolytic treated WT mice.
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challenged, Muc5ac _/" mice, which have previously been shown to be protected against AHR in response to AOE challenges and MCh administration.
Histology and Western Blots
Airways of a healthy, non-AOE challenged BALB WT mouse tended to show patent airways free of mucus plugs. (Fig. 16). This image shows a bronchus (a large central airway) that is completely free of mucus. The airways in these mice also appeared to have no airway thickening. The AOE challenged, non-mucolytic treated BALB WT mice were prone to have mucus plugs that often occluded the larger airways (Fig. 17). This image shows a bronchus that almost totally occluded by a mucus plug with only a small airspace left. A situation such as this would prevent air from reaching all airways distal to this occurrence. This could be potentially an entire lobe of the lung.
In regards to the AOE challenged BALB Muc5ac _/" mice, airways tended to display some degree of mucus secretory cell metaplasia, but airways usually remained patent (Fig. 18). Some remaining mucus (Muc5b) is present in this image but it appears to remain in the secretory cells.
The microscopic appearance of an AOE Challenged, P-3001 treated WT mice usually showed patent airways, but there was often some mucous cell metaplasia (Fig. 19). As with the BALB Muc5acmouse, the mucus appears to remain confined to the secretory cells.
This may be the result of mucociliary clearance of secreted mucus with the remaining signal resulting from residual intracellular mucin present in the treated mice.
To confirm that P-3001 affected the mucin targets as predicted, Western blotting was performed under non-reducing conditions. With the Muc5b Western blot, the mucin polymers have been effectively broken down (low molecular weight) in the P-3001 treated
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Figure 16. The patent airway of an unchallenged WT mouse. Airways in an unchallenged mouse display the absence of airway thickening and excessive mucus (arrow).
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100 \i m
Figure 17. Non-mucolytic treated AOE challenged WT mouse with mucus airway obstruction. Airways show mucus in a bronchus (black arrow) and marked mucus airway occlusion in the bronchiole (yellow arrow).
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1
Figure 18. Absence of mucus plugging in a Muc5ac mouse. Histology showing the airway of an AOE challenged BALB Muc5ac mouse. Note the airway thickening in the absence of mucus plugging (arrow).
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Figure 19. Absence of mucus plugs in a P-3001 treated WT mouse. AOE challenged-mucolytic treated mouse showing airway thickening with the absence of mucus plugging {arrow).
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WT mice (Fig. 20). In contrast, the mucin proteins of the AOE challenged, non-mucolytic treated WT mice have a higher molecular weight, indicating that the polymers are still intact.
Discussion
Asthma is a serious and sometimes fatal disease that affects 25 million people in the United States alone. Asthma is characterized by airway inflammation, smooth muscle contraction, and mucus hypersecretion that contribute to AHR. The current strategies in the treatment of asthma target the control of inflammation and smooth muscle contraction, crediting them as being the foremost causes of airflow obstruction. However, even though mucus hypersecretion and mucus plugging have been noted to be key features of mild-to-moderate disease and to fatal asthma attacks, these components are poorly resolved. A lack of therapeutic options stems from an incomplete understanding of the mechanisms of mucin-mediated obstruction.
Under normal conditions, mucin glycoproteins are important for airway defense. However, in diseased states, mucus hypersecretion can be life-threatening (Thornton et al. 2008). Mucin proteins are large and complex structures that should ideally be studied in vivo but there are few available models (Thornton et al. 2008). Consequently, research on novel pharmacologic treatments for mucus remains deficient but nascent. Recent literature has identified a specific polymeric mucin protein (Muc5ac) as being responsible for airway hyperreactivity in mice (Evans et al. 2015). Although mucus plugging features prominently as the cause of death, mucolytics are not currently being added to treatment regimens. As previously noted, this is due, in part, to the lack of options available to clinicians.
Dornase alfa, a mucolytic currently on the market, works by breaking up the
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Figure 20. Mucin Protein breakdown in P-3001 treated WT mice. Western blot on bronchoalveolar lavage fluid (BALF) samples from saline and P-3001 treated WT mice. Results showed mucin glycoprotein breakdown in the mice that were treated with the mucolytic P-3001.
48


extracellular DNAbut not the mucin glycoproteinsin a patients mucus (Gardenhire and Rau 2008). This is particularly effective for patients with cystic fibrosis, where mucus is typically purulent (Gardenhire and Rau 2008). However, in cases of moderate to severe asthma Dornase alfa is not an effective mucolytic, most likely because the relative amount of DNA in asthmatic compared to CF mucus is too low for the drug to be effective (Boogaard et al. 2008).
Distinct targets for mucolysis that target mucin glycoproteins are the cysteine-rich domains that are responsible for disulfide bond-mediated polymer formation. One of these N-acetylecysteine (NAC, Mucomyst), is available on the market. However, it is very inefficient and must be given to patients at very high concentrations. Accordingly, an unfortunate side effect of NAC is bronchospasm, which is to be avoided in people with asthma, since bronchospasm is a manifestation of asthma. Future mucolytic compounds need to address the presence and viscosity of mucus, with higher efficacy at lower concentrations that do not cause airway contractility.
Furthermore, new mucolytic drugs need to preserve Muc5b, as diminished amount of this mucin can result in increased mortality from respiratory infections (Roy et al. 2013). Having identified a target in airway mucus (Muc5ac), it is reasonable to assume that research can proceed to develop a novel pharmacologic compound to treat mucus occlusion in asthma, and potentially decrease asthma fatalities. As noted above, hypersecretion of Muc5ac can be problematic, whereas the production of Muc5b is necessary. Thus, future pharmacologic compounds need to diminish Muc5ac while preserving Muc5b. Additionally, asthma treatments should avoid any potential adverse effects such as bronchospasm or rapid liquification of airway secretions, as these events can occlude the airway.
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Although contractile forces are a contributing cause to airflow obstruction in asthma (An et al. 2007), contractile forces alone are not adequate to explain the total airway occlusion noted in fatal asthma attacks. Mucus plugging features prominently in fatal cases of asthma (see Figs 1, 2, and 3). In a study by Kuyper et al. (2003), 275 airways from 93 cases of fatal asthma were examined. They found that mucus occlusion ranged from 4% to 100%, but only 5 cases showed less than 20% occlusion (Kuyper et al. 2003). This indicates that further research into the use of mucolytics in the treatment of asthma is appropriate. My work illustrates the efficacy of a phosphine based mucolytic compound in the treatment of a mouse model of allergic asthma by reducing the viscoelasticity of mucus.
My study employed techniques in mice that elicited an allergic response using an inhaled allergen and MCh delivery. I adapted existing methods in the laboratory by employing IV administration of MCh (the majority of previous studies have delivered MCh by aerosol). MCh was given IV in order to separate its delivery routes from the therapeutic mucolytic test compound P-3001, so as to prevent competition of medications. The phosphine reducing agent P-3001 is capable of dissolving disulfide bonds of mucin proteins, thereby decreasing the viscoelasticity of mucus. Theoretically, this allows for improved mucus clearance, lessening the incidence of mucus plugging associated airflow obstruction.
Indeed, AOE challenged WT mice that received aerosolized P-3001 showed decreased Rrs changes in response to IV MCh than AOE challenged non-mucolytic treated WT mice. The mucolytic treated, AOE challenged WT mice showed a lower respiratory system resistance compared to the AOE challenged untreated mice. Linear regression on log (x) transformed data showed that the slopes of the AOE challenged untreated WT mice were statistically significant (p-value < 0.05) by one-way ANOVA and Dunnetts multiple
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comparison tests to all other groups. This confirms that P-3001 was protective against AHR in these mice. The Rrs levels of the treated mice were similar to the MucSac _/" mice, which have previously shown to be protected against AHR. Importantly, given the striking overlap between these two findings, my data suggest that the mucolytic effect induced pharmacologically (acute reversal) is as efficacious as complete absence of Muc5ac driven by gene deficiency (genetic prevention).
Given that my data show that the mucolytic P-3001 is an effective reducing agent in a mouse model of allergic asthma in mice, it is enticing to speculate that future treatments could employ the use of a mucolytic compound in the treatment of asthma in humans. My results show that not only were the mice protected against AHR, but also demonstrated the breakdown of mucin proteins with western blots, and decreased mucus plugging illustrated by histology. Mucolytics may prove to be a useful compound to recruit in the treatment of human asthma.
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CHAPTER III
FUTURE DIRECTIONS
Future studies with mucus altering compounds will necessitate an accurate method of mucus quantification. One possibility is to use microscope imaging of histology for mucus quantification. Another approach would be the use of carbon nanoparticles delivered by aerosol. The use of fluorescing as well as black carbon nanoparticles were attempted in this study. The techniques of both methods will need to be refined further to elicit reliable, unbiased data.
Microscopy Methods
The following procedure was employed for this purpose. Slides were initially loaded into microscope and the overview area was defined to include all stained lung sections. In order to attain a random sample, each section from the overview was initially visualized at 4 times magnification specimen objective. Field of vision was arranged to be the approximate center of the tissue, with image unfocused, so that no structures could be identified. Magnification was then increased to 10 times and the image was taken. There were approximately 45 images per slide. Two duplicates of each image were created to facilitate counting of both, airway versus parenchyma, and the presence or absence of mucus in the bronchi and bronchioles.
Using a digital reticle, a grid of precise density overlaid on images, areas of airways and extracellular mucus were quantified using count and measure tools in the cellSens software (Olympus, Center Valley, PA). For the airway versus parenchyma counts, a 250 gm/250 pm grid was overlaid on image (Fig. 21). Intersections of the grid were declared
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Figure 21. Airway versus Parenchyma grid for mucus quantification. Initial counts of airway versus parenchyma were done using the digital reticule and count and measure features on the Olympus BX63 microscope. A 250/250 grid superimposed on image showing counts for parenchyma (blue crosses), and airway (green crosses).
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either airway, marked with green crosses, or parenchyma, marked with blue crosses. The cellSens software maintains a tally of the percentages of each class.
Regions that were denoted airway in the above grid, were given a closer inspection to assess the amount of mucus present. These images were fixed with a 50 pm /50 pm grid and counts were made in the region containing the airway. Intersections that contained mucus in bronchi were labeled with the red cross and airway lumen in this region was labeled with a purple cross. Intersections that contained mucus in a bronchiole, were labeled with a salmon cross and airway lumen in this region was labeled with a yellow cross (Fig. 22). Terminal bronchioles and smaller airway/alveolar spaces were not tallied as previous investigations have revealed that they are not lined by mucin-producing cells and thus rarely contain any secreted mucus.
Microscopy Results
Marks are then quantified to relative percentages. The quantification results of an AOE challenged, untreated BALB WT mouse (n = 1), and that of the AOE challenged, P-3001 treated BALB WT mouse (n = 1) are shown (Table 2). Results show that the AOE challenged, non-mucolytic treated WT mice had 46% occlusion in the bronchi, versus only 16% occlusion in the P-3001 treated WT mice. The combined counts for airway occlusion for the bronchi plus bronchioles showed that 25% of the airways were occluded with the challenged untreated mouse, compared to the P-3001 treated who only had 10% occlusion. Also of note, the P-3001 treated mouse had a greater percentage of mucus in the bronchi (16%) versus in the bronchioles (3%). This may indicate that mucociliary clearance was effectively mobilizing mucus toward the larger airways for clearance.
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Figure 22. Airway versus mucus quantification grid. Image showing a 50/50 grid used for quantification of airway mucus in images previously identified to contain an airway. Intersections with mucus in a bronchi were labeled with a red cross, bronchi with clear airway lumen were labeled with a purple cross. Intersections with a bronchiole containing mucus were labeled with a salmon cross, and bronchiole clear airway lumen with a yellow cross.
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Table 2. Quantitative assessments of mucus plugging for an AOE challenged, untreated
mouse and an AOE chal enged, P-3001 treated mouse.
Total Lung AOE Challenged Untreated 912 Total Counts AOE Challenged + P-3001 432 Total Counts
Airway 61/912 (6.7%) 44/432 (10.2%)
Parenchyma 881/912 (93.3%) 388/432 (89.)8%

Lumen vs Mucus AOE Challenged Untreated 781 Total Counts AOE Challenged + P-3001 779 Total Counts
Bronchi Positive 82/177 (46%) 71/453 (16%)
Bronchi Negative 95/177 (54%) 382/453 (84%)
Bronchiole Positive 116/604(19%) 10/326 (3%)
Bronchiole Negative 488/604 (81%) 316/326 (97%)
Combined Bronchi & Bronchiole Positive 198/781 (25%) 81/779(10%)
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Another interesting finding is that the percentage of airways is lower for the untreated mouse in the airway versus parenchyma count. Since the total number of larger airways does not change, this may indicate an increase in parenchymal airspaces (hyperinflation from airflow obstruction) making the airway percentage smaller relative to the parenchymal airspaces. This is a developing system and the technique is being refined. The end goal of this procedure is to have an unbiased, random, and efficient method of mucus quantification. Carbon Nanoparticle Methods
Fluorescent carbon nanoparticles are emerging as a useful labeling technique, and have shown superior brightness and photostability compared to conventional molecular probes (Bhunia et al. 2013). Initial research into the use of nanoparticles to assess mucociliary clearance have shown that mucociliary clearance of particles is independent of size, shape and charge of the particles, probably due to the lack of penetration of particles through the mucus blanket (Kirch et al. 2011). Nonetheless, the use of aerosolized nanoparticles is of current interest for both diagnostic and therapeutic research (Yang et al 2008).
The use of fluorescent and black carbon nanoparticles was explored with this study, but results were mixed. Carbon nanoparticles were employed with intubated mice. Particles were delivered, and mucociliary clearance was assessed at different time points (time 0-60 minutes) using bronchoalveolar lung fluid (BALF). After samples were obtained, they were scanned with a Bio Tek plate reader (Bio Tek Instruments, Winooski, VT). I was unable to ascertain any significant differences with this technique, as all samples failed to yield signals above the autoflourescence of lung lavage fluid. It is unclear if the lack of signal was due to
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aerosol delivery, low yields from lavage, or weak fluorescence of the particles (<10% quantum yield).
In addition to the above techniques, black carbon nanoparticles were also investigated using the same surgical procedures previously described (see methods, Chapter II). In a pilot study, observed robust deposition of particles (Fig. 23). However, since the particles were non- fluorescent, I sought to determine their localization and transport by microscopy. Mice were ventilated and treated with saline or P-3001 and MCh exactly as described for the AHR studies above (again, see Methods). Delivery of black CNP particles was accomplished with the use of the flexiVent. 150 nm black CNP were diluted to 25 mg/ml to facilitate aerosol delivery. Animals received six 10 second doses for a total delivery of 4.945 mg of particles.
Mice were then euthanized and lungs fixated in methacarn and prepared for histology as before. Initial results have shown that black carbon nanoparticles are visible on the airway surfaces before being embedded in paraffin. Unfortunately, after the paraffin embedding and staining processes, nanoparticles are largely absent on the slides. This may be due to the rehydration/dehydration process with xylene and graded alcohols that the slides undergo during the staining process, as well as the poor adhesion of the neutrally charged particles on the positively charged glass microscope slides. Further experiments will help to refine a technique that will mark the progress of mucociliary clearance. Perhaps other methods such as the use of Ulex Europaeus Agglutinnin I (UEA I), a lectin that binds to the glycoproteins on the mucins, could be used to mark mucociliary clearance (Roney et al. 2011). Finding a useful technique that is random, unbiased, and reliable in the laboratory setting would be
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Fig. 23. Left lung filet illustrating deposition of black carbon nanoparticles (CNP) at time 0.
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helpful to assess the efficacy of pharmacological compounds on improving mucociliary transport in the future.
Conclusions and Impact of Findings
In summary, using a mouse model of human asthma, I have demonstrated that a novel mucolytic compound P-3001 was capable of reversing AHR and mucus plugging in mice via disruption of mucin polymers. I have also shown that reversing mucus hypersecretion is a potential therapeutic strategy for allergic asthma. As new research regarding mucin proteins is emerging, concurrent research in pharmacology should be undertaken. Since mucus is prominent in asthma episodes and a key feature of fatal asthma attacks, research needs to be continued using new drugs, specifically mucolytics. As noted by Sidebotham and Roche in 2003, asthma deaths are a persistent and preventable mortality.
The use of a phosphine based reducing agent could be an effective therapeutic strategy for reducing the viscoelasticity of mucus in the airway, thereby, reducing plugging. Hence, phosphine based reducing agents could be effective in reducing fatalities as well improving quality of life for patients with mild to moderate disease. Mucolytics may also prove useful for other diseases with impaired mucociliary clearance such as, COPD, CF, and ciliary dyskinesia.
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Zdanowicz, M. M. (2007). Pharmacotherapy of Asthma. American Journal of Pharmaceutical Education, 77(5), 98. doi:10.5688/aj710598
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APPENDIX A
Protocols for FlexiVent and Western Blots
Protocol for flexiVent
FlexiVent In Vivo Asthma Study Protocol With IV Methacholine
1:1 day to 1 week prior to experiment
Click on flexiware icon to launch welcome dialog
Welcome dialog -^Study Definition and Planning
Create new study File menu *New study or click create a new study
button. Fill in the required info and click next until you reach step 6:
confirmations.
1. Title and status: Assign the study a title, enter a name of the person responsible for the study and select a status for the study (Identified or Scheduled) Next
2. Objective and Hypothesis: Next
3. Protocol:* Next
4. Subject groups:* New button Subject group dialogue Enter the title of your group, status (started or not started), subject quantity in the group and details OK* Next
5. Assign subject: To assign subject Subject management button *
Create new subject icon * Subject Detail Dialogue* Fill in the
unique subject identifier, species and the subject weight must be completed; the remaining fields (e.g. strain, birthdate) are optional assign the subject to group membership (found below subject properties)* select subject group statues (valid/invalid or pending)* OK* Next* Finish.
6. Confirmation
Supplies needed for experiment:
1 x18 gauge beveled blunt tip cannula /mouse 1 piece of silk or polyester thread tie (15-20 cm long)
1 ml syringes (urethane, succinylcholine for i.p. bolus, methacarn, saline flushes for IV insertion, and to collect BAL)
3 ml syringe for IV infusion of succinylcholine and methacholine
Tubes for BALF collection (6 x 1.5 ml, 1 x 1.0 ml per mouse, 2 cryovials per mouse)
Scintillation vials filled with methacarn for fixation (1 per mouse)
20 gauge shielded IV catheter to cannulate the inferior vena cava Saline
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succinylcholine chloride (0.2 ml of a 20 mg/ml solution) for i.p. injecion and (50 gg/ml) for IV infusion
methacholine (5 gl/g dose of a 20 gg/ml solution) added to succinylcholine for IV infusion urethane (2g/kg of a 0.01 ml/g solution) for i.p. injection surgical instruments p 200 and p1000 pipettes and tips
II: On the day of experiment
Prepare the styroform setup: stryrofornw chemical heating pads (one for each mouse) -^two paper towels and tape for securing mice and platform.
Fill and prime 3 ml syringe with succinylcholine chloride and methacholine mixture
Prepare supplies for IV insertion
Set up syringe mini pump (rate 4 pl/g/min)
Prepare syringes for i.p. urethane and succinylcholine administration, saline for IV flushes, and methacarn for fixation
Fill scintillation vials with methacarn and label, then place on ice
Cut lengths of silk thread (one for each mouse)
Flexiware Welcome dialogue Experimentation session
At this point the program will prompt you with series of dialogues and wizard
> Study selection : Select your study that you predefined and saved
> Template properties : Mouse (AN/EKG) custom OK
> Flexiware module. 1,55mlOKwerify the module dialogue (flexivent FX1) OK
> Add module with aeroneb mount
> Attach aeroneb
> Subject site assignment, drag and drop the animal that you going to use from right box to leftOK
> Subject weight confirmation, weigh the mouse and enter the current weight
> Auto- calibration and warm up
> Prime the nebulizer, connect the nebulizer Place 100 pi Saline hold it up and click next you should see aerosol emerging from the bottom of aeroneb, and when finish clean excess PBS and reattach to base
Calibration:
Make sure that each tube and nebulizer is dry, and there are no leaks. Channel calibration:
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Step 1: (Channel Selection) both Cylinder pressure (step 2-5) and Airway pressure (step 6-9) boxes are checked next Cylinder pressure calibration
Step 2: First value: Zero Atmosphere (0 mmhbO): Leave the Y-tubing open to atmosphere ->NEXT
Step 3: Second Value: Known Pressure: Connect the SCIREQ manometer to the Y-tubing apply pressure over 300 mmhhO mark via syringe (The indicator panel on the right pressure bar will turn from red to green) > Click next when water from manometer reach 300 mmhhO (**this value can be enter if you click next at other pressure value other than 300 mmhhO.
Step 4: Target value input: Enter 300 mmhhO next
Step 5: Channel calibration results: ranges should be Max value = 80 8 cmhhO and Min value = -80 8 cmhhO, range 160 16
Airway pressure calibration
Step 6: First value: Zero atmosphere (0 mmhhO): Leave the Y-tubing open to atmosphere ->NEXT
Step 7: Second Value: Known Pressure: Connect the SCIREQ manometer to the Y-tubing apply pressure over 300 mmhhO mark via syringe (This will indicator panel on the right pressure bar will turn from red to green > Click next when water from manometer reach 300 mmhhO (**this value can be enter if you click next at other pressure value other than 300mmH20.
Step 8: Target value input: (Enter known value for the first point) > next
Step 9: Channel calibration results: ranges should be Max value = 80 8 cmhhO and Min value = -80 8 cmhhO, range 160 16
Click Finish to exit channel calibration
Tube Calibration: Should be performed before EACH new subject in an experimentation session Stepl: Welcome dialog^ next
Step 2: Perturbation selection: TLC, Snapshot-150 and Quick Prime-3 are to be selected ->NEXT
Step 3: Closed Preparation: Place tracheal cannula on ventilator. Block the tracheal cannula with your thumb. next
Step 4: Closed Calibration: Keep cannula blocked while the system performs closed calibration.
Step 5: Open Preparation: Remove thumb from the tracheal cannula so it is open to the air NEXT
Step 6: Open Calibration: Keep cannula open while the system performs open calibration.
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Check values: Prime TLC Rs (shunt resistance) > 2000
Snapshot (tube resistance) should be lower that baseline resistance 0.38
cmhhO
Once the calibrations are done, you will be prompted to begin the default ventilation. Do not start ventilation yet. First, perform surgery on mouse.
Animal Preparation and Performing the Experiment
Anesthetize first mouse 10 pl/g of urethane (concentration: 200mg/ml dosage; 2.0g /kg)
To tracheostomized:
Remove strap muscles to expose trachea
Separate esophagus away from trachea using forceps
Slip thread under the trachea as a half loop
Cut the thread in the middle leaving two equal size pieces
Make a small cut (~0.1 in) on the trachea with micro-scissors
Slip in cannula (half its length inside the trachea) and tie the thread in order to secure cannula.
Start ventilation
Connect tracheal cannula to ventilator, while keeping cannula and mouse in the neutral position above the heating pad, then secure mouse to the platform Inject mouse with 0.2 ml succinylcholine chloride (10 pg/ml) i.p.
Open the abdomen and identify the inferior vena cava (IVC)
Cannulae the IVC with the 20 gauge sheilded IV catheter and confirm success by observing blood return in the catheter
After flushing the catheter with saline, connect IV tubing insuring that there are no air bubbles in the line
Start the syringe pump at rate of 4 pl/kg/min.
Check for adequate paralysis. Look at the monitor panels on the bottom of flexivent screen-look for even peaks and valleys.
Load Script:
Automation > load scripts > select script (e.g. Leslie-Inhaled dose response with IV infusion) > double click script name in window or select alternate start with automation start script
Begin with the baseline (4 pl/kg/min of methacholine), when prompted
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Doses of IV succinylcholine and methacholine mixture:
4 |jl/kg/min 8 gl/kg/min 16 gl/kg/min 32 gl/kg/min 64 gl/kg/min 128 gl/kg/min
- ***ALLOW FULL RECOVERY BETWEEN DOSES***
Once you finish all doses, click cancel on the continue with next dose? dialogue box Stop default ventilation>stop recording select flexivent > stop or ctrl-O Stop the syringe pump
Remove mouse from flexivent and proceed with sample collection.
If subject is to be prepared for histology, remove cannula and immediately tie off trachea Inject 0.4ml of methacarn into the intrapleural space and gently distribute by rotating After 20 minutes, excise heart and lungs from the chest cavity and place in the scintillation vial filled with methacarn Place specimens in the 40 C refrigerator Between mice, the following should be performed
Detach nebulizer and Y tubing from their base and dry condensation with Kimwipes.
Blow compressed through the Y tubing, to flush out any remaining condensation.
Reload succinylcholine and methacholine for next mouse in the syringe pump.
To start the next animal Edit assign subject and load the next mouse to subject assignment
Start channel calibration
o Closed Preparation: Place tracheal cannula on ventilator. Block the tracheal cannula with your thumb. next
o Closed Calibration: Keep cannula blocked while the system performs closed calibration.
o Open Preparation: Remove thumb from the tracheal cannula so it is open to the air next.
o Open Calibration: Keep cannula open while the system performs open calibration, o Verify the values Rrs value
Clean Up
Remove the Y- tubing adaptor.
Connect a quick connect to the expiratory port on the front of the module (right).
Place a second quick-connect in the air/gas exhaust port on the back of the module (bottom) Place a beaker at the exit of the tubing connected to air/gas exhaust port.
Using 10 ml syringe, run 6 x 10 ml isopropyl alcohol into the tubing that is attached to the expiratory port.
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Repeat previous step as needed.
Flush out any liquid remaining in the lines by blowing air through the tubing attached to the front expiratory port.
Clean Y tubing with water and dry with kimwipes Remove quick connects.
Replace Y tubing adapter
Attach the test load (1 ml syringe) to Y tubing
Start default ventilation and let it run for approximately 15 min to ensure that the expiratory line is completely dry.
After 15min, stop default ventilation Close program
Reviewing data/Export
Welcome dialogue Reviewing and reporting select the study you wish to view Tools Option New Name of scenario Data type- event parameters Parameters +signals(G,H,COD, Rrs, Crs, Ers) next Format Excel Finish Apply OK File Export Export data tick the box next Export frames entire frame mext export destination desktop export
Data export
-Data export is divided into two portions in flexiWare: Export Scenarios and Data Export.
Export Scenarios are containers in which an export strategy is described. They are generic and define only the type of data to be exported (e.g. subject data, parameters) and the export type (e.g.ASCII, Excel). They are saved in the database and may be used repeatedly.
Data Export delimits the actual data to be exported (e.g. subject selection, timeframe).
EXPORT SCENARIO
- To define a new Export Scenario, select Tools > Options> Export scenarios
- From the Export Scenarios screen new button
-You must provide a unique name, the type of data you wish to export in this scenario and for flexiVent applications only, the relevant perturbations. There are four types of data that can be exported:
> Subjects: exports all of the subject details including statistics regarding data collection Completed with the subject.
> Dataset Parameters: exports analyzed parametric outcomes from datasets.
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> Dataset Signals: exports analyzed non-parametric outcomes from datasets; these are often intermediary results which are used to generate the final dataset parameters.
> Stream Data: Exports continuous streams of data
- When selecting dataset parameters or dataset signals, you must enter the name of the data to be exported. The names entered must match the names that are defined for each of the outcomes on the applicable analyzer page(s) of the Perturbation Properties dialogue.
-Specify whether you wish to export the data to Microsoft Excel or ASCII format. This screen also gives you the opportunity to include excluded datasets, event comments and user markers
-Click Finish.
DATA EXPORT
-Once you have created your export scenario(s), you can proceed to data export by selecting >File > Export > Export Data. This displays the Data Export Wizard that allows you to export your data in five easy steps.
1. Select the Export Scenario(s) you would like to use. You can export data using multiple data export scenarios simultaneously.
2. Select the subject(s) for which you wish to export data. The subject filter at the
top of the screen allows you to select All subjects, those marked as Valid, those marked as Invalid or Selected subjects. If you choose Selected subjects, you can multi-select by holding down the Ctrl key as you click on each subject.
3. Specify the export timeframe for data export. The following three choices are available:
> Entire study timeframe exports all data belonging to the current study.
> Between specific events allows you to select a start and end event.
> Based on subject time allows you to export between specified times using the relative time scale i.e. all subjects start at time 0, for example, this option would allow you to export the first 10 minutes of data for each subject.
4. Specify the export destination folder. If you leave it set to the default, your data is exported to C:\Users\Public\Documents\SCIREQ\flexiWare\[Study Name], The file(s) from the export will be named according to the selected export scenario. On this screen you can also specify how the software should proceed if filename for the exported data already exist.
5. Click Export
Protocol for Western Blots
Western Blot Protocol for Mouse Muc5b from BALF Licor Materials needed:
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LE Agarose Gene Mate Cat No. E-3120-500
Gel unit (Recycling B3 unit or the Large A series unit from the Lozupone Lab)
Gel/Running Buffer (2L)-1XTAE +0.1% w/vSDS (add 1600ml H20 +40ml 50X TAE -> bring to 2L -> mix -> take out 20ml of solution ->+20ml 10% SDS -> mix well)
Glassware: cleaned well using Sparkleen -> rinsed in Dl Water
PVDF Membrane: IPFL00010 Immobilon-FL PVDF, 0.45 pm (membrane cut to 125 x 140 mm)
100% MeOH
Paddle tweezers (2 pairs)
Pencil to mark membrane
Sereological pipette 5 mL to roll out gel
Vacuum flasks: with in H2O vacuum guage(s) and regulator(s)
Blot Paper: BIO-RAD thick blot filter paper (un-cut) Cat no. 1703955
DTT:- Amresco CAS Number: 3483-12-3 (dry powder in -20C)
Odyssey Blocking Buffer (TBS), 125 mL [P/N 927-50100]
Working Solutions: 50% Odyssey blocking solution and 50% 1X TBS for blocking or 50% 1X TBST for the antibody dilutions/incubations (~40 ml will cover blots)
1 Ab: rabbit-anti-mouse Muc5b (-20C aliquoted undiluted Ab; use at 1:5,000)
2 Ab: IRDye 680RD Goat-anti-Rabbit (Lot# C41029-03; Stored undiluted at 4C; used at 1:15,000 dilution)
1 X TBS: 12.114 g Tris-HCI + 87.66 g NaCI, pH 8.0 and make up to 1 L with ddH20
1 X TBST: 1 x TBS + 0.1 % Tween-20
10% SDS stock solution
20X SSC: 175.32g NaCI + 88.2g Na Citrate, fill to 1L with dd H2O, pH to 7.0
Working solution (per liter): 4x SSC: 200 ml of 20x SSC + 800 ml ddH20
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1X TBS: (12.114 g Tris-HCI + 87.66 g NaCI, Adjust pH to 8.0 and make up to 1 L with ddH20)
1 X TBST. 1x TBS + 0.1% Tween-20
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Plate well Offest vol. BAL vol Sample ID
fnntinnah______________________________
Gel Preparation Template:
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18




Optimization Tips (from LI-COR)
1. To avoid background speckles on blots, use high quality ultrapure water for buffers.
2. Rinsing previously-used incubation boxes with methanol can reduce background contamination of future blots
3. Never perform incubations or washes in dishes that have been previously used for Coomassie staining.
4. Membranes should be handled only by their edges, with clean forceps
5. Always pour-off antibody solution and washes from the same corner of the box to ensure complete removal of previous solutions.
6. After handling membranes that have been incubating in antibody solutions, clean forceps thoroughly with Dl water and/or methanol, then rinse with distilled water.
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7. Do not wrap the membrane in plastic when scanning.
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Procedures:
WESTERN BLOT DAY 1
Prepare running buffer and cool in the cold room.
Make 0.9% agarose 0.1% SDS TAE 14x13 cm gel:
Weigh (record weight) and tare 500 mL flask (use high wt capacity balance)
Add 100 mL ddH20 + 1.62g agarose to flask
Cover flask opening with KIM wipe to avoid spill over
Boil: set timer on the microwave to 3 min -> let run for 1 min -> stop and gently shake -> stop every 20-30 sec thereafter
Cool for a couple of min
Heat additional 100 ml of ddH20 for ~1 min in microwave
Add 3.6 mL 50x TAE and 1.8 mL 10% SDS (along the glass to prevent bubbling)
Reweigh (record weight to make sure appropriate volume lost is added back; 1 mL H20 = 1 g)
Fill with hot ddH20 until 180 g
Use 1.5 mm 20-well comb side to cast the gel
Thaw BAL samples in ice/water slurry. (Add protease inhibitor, 10% of sample volume.)
Label 1,5ml tubes with arbitrary numbers (e.g., 1,2,3...)
Swirl BAL samples with pipette tip, and add BALF and H20 to labeled 1.5 ml tubes according to the normalization analysis for the total of 25 ul.
Add 5 ul of loading buffer (nominally 10X buffer)
Spin down the tubes for briefly (3-5 sec) on benchtop microfuge
Fill gel unit with running buffer.
Load gel (DO NOT use 2 outside lanes: these will not transfer).
Run overnight at 42 V in cold room or deli cooler.
WESTERN BLOT DAY 2
Photograph gel with a ruler to see marker location
Soak gel in 4x SSC + 10mM DTT (0.15425g DTT per 100mL of 4x SSC) at RT for 20 min with gentle shaking (10-20 RPM)
Activate PVDF FL in 100% MeOH for 15 sec
Dump MeOH and add 50-100 ml 4x SCC.
Gently shake (10-20 RPM) >5 min.
Assemble transfer apparatus: sponge -> filter paper
Saturate filter paper with 4X SSC prior to placing PVDF membrane on top
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Finish assembling the transfer apparatus -> PVDF membrane (roll out any visible bubbles with serological pipette and keep membrane wet with 4x SSC), transfer mask, gel.
Once the gel is in place make sure there are no bubbles underneath by rolling them out with a stereological pipette.
Turn on vacuum and set to 10-15 inches FI2O
Pour 4X SSC to submerge the gel.
Vacuum for 90
Make sure there are NO leaks! Do not leave the transfer unit running unattended.
Remove PVDF membrane with clean tweezers and rinse for 2-3 in 4XSSC
Do not let PVDF to dry
Place membrane in incubation box and block the membrane in Odyssey Blocking buffer working solution for 1 hr on rocker (set rocker to 1-2)
Prepare 1 antibody dilution
o 20 ml Odyssey blocking buffer o 20 ml TBST
o 8 uL 1 Ab rabbit-anti-mouse Muc5B o Mix well
o Add to the membrane
Incubate ON @ 4C with gentle rocking (dial on 4C rocker at 10-11 oclock).
WESTERN BLOT DAY 3
Bring membrane to RT.
Wash membranes
o Pour off primary antibody solution o Rinse membrane with IX TBS-T (0.1% Tween 20), dump immediately
o Cover blot with 1X TBS-T (0.1% Tween 20) o Rock for 5 (set rocker to 1 -2) o Pour off wash solution o Repeat for a total of 3-4 washes
Prepare 2 antibody dilution
o 20 ml Odyssey blocking buffer o 20 ml TBST
o 40 ul 10% SDS stock solution (final cone. =0.01%) SDS) o 2.7 ul Secondary Antibody (1:15,000) o Mix well
o Add to the membrane
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Protect the membrane from light during incubation using box or foil.
Incubate @ RT for 1 hr with gentle shaking Protect membrane from light during washes!
Wash membranes
o Pour off primary antibody solution o Rinse membrane with 1X TBS-T (0.1% Tween 20), dump immediately
o Cover blot with 1X TBS-T (0.1% Tween 20) o Rock for 5 (set rocker to 1 -2) o Pour off wash solution o Repeat for a total of 3-4 washes
Rinse membrane with 1X-TBS to remove residual Tween 20.
Membrane is now ready to be imaged
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APPENDIX B
FlexiVent Raw Data
Group Dose Rrs
Uncha lenged, n = 7 4 1.697806
Uncha lenged, n = 7 4 1.485296
Uncha lenged, n = 7 4 1.598057
Uncha lenged, n = 7 4 0.738117
Uncha lenged, n = 7 4 1.21963
Uncha lenged, n = 7 4 0.841489
Uncha lenged, n = 7 4 0.843196
Uncha lenged, n = 7 8 1.613589
Uncha lenged, n = 7 8 1.770998
Uncha lenged, n = 7 8 2.127709
Uncha lenged, n = 7 8 0.818583
Uncha lenged, n = 7 8 1.018494
Uncha lenged, n = 7 8 1.032738
Uncha lenged, n = 7 8 1.956596
Uncha lenged, n = 7 16 1.626127
Uncha lenged, n = 7 16 1.767881
Uncha lenged, n = 7 16 2.008483
Uncha lenged, n = 7 16 0.869276
Uncha lenged, n = 7 16 1.14151
Uncha lenged, n = 7 16
Uncha lenged, n = 7 16 2.467539
Uncha lenged, n = 7 32 2.585368
Uncha lenged, n = 7 32 1.929785
Uncha lenged, n = 7 32 2.973254
Uncha lenged, n = 7 32 0.930825
Uncha lenged, n = 7 32 5.405394
Uncha lenged, n = 7 32 2.190499
Uncha lenged, n = 7 32 2.974648
Uncha lenged, n = 7 64 4.002827
Uncha lenged, n = 7 64 2.280709
Uncha lenged, n = 7 64 3.689791
Uncha lenged, n = 7 64 0.977061
Uncha lenged, n = 7 64
Uncha lenged, n = 7 64 3.036626
Uncha lenged, n = 7 64 3.049514
Uncha lenged, n = 7 128 5.931221
Uncha lenged, n = 7 128 2.578164
Uncha lenged, n = 7 128 3.689791
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Unchallenged, n = 7 128 1.067326
Unchallenged, n = 7 128
Unchallenged, n = 7 128 3.138034
Unchallenged, n = 7 128 4.626993
BALB WT AOE, n = 13 4 1.573098
BALB WT AOE, n = 13 4 1.797719
BALB WT AOE, n = 13 4 1.839151
BALB WT AOE, n = 13 4 1.216133
BALB WT AOE, n = 13 4 1.546626
BALB WT AOE, n = 13 4 1.227731
BALB WT AOE, n = 13 4 1.484908
BALB WT AOE, n = 13 4 1.471928
BALB WT AOE, n = 13 4 1.454716
BALB WT AOE, n = 13 4 1.80948
BALB WT AOE, n = 13 4 1.509645
BALB WT AOE, n = 13 4 1.225105
BALB WT AOE, n = 13 4 1.880144
BALB WT AOE, n = 13 8 0.912943
BALB WT AOE, n = 13 8 3.739564
BALB WT AOE, n = 13 8 2.645449
BALB WT AOE, n = 13 8 1.472816
BALB WT AOE, n = 13 8 1.327337
BALB WT AOE, n = 13 8 0.773229
BALB WT AOE, n = 13 8 1.779645
BALB WT AOE, n = 13 8 4.652586
BALB WT AOE, n = 13 8 4.785667
BALB WT AOE, n = 13 8 2.144764
BALB WT AOE, n = 13 8 3.641887
BALB WT AOE, n = 13 8 1.153776
BALB WT AOE, n = 13 8 2.264587
BALB WT AOE, n = 13 16 1.355516
BALB WT AOE, n = 13 16 3.70446
BALB WT AOE, n = 13 16 1.839151
BALB WT AOE, n = 13 16 9.660468
BALB WT AOE, n = 13 16 1.744475
BALB WT AOE, n = 13 16 0.901226
BALB WT AOE, n = 13 16 4.222582
BALB WT AOE, n = 13 16 3.119045
BALB WT AOE, n = 13 16 9.653567
BALB WT AOE, n = 13 16 8.163266
BALB WT AOE, n = 13 16 11.67767
BALB WT AOE, n = 13 16 2.09324
BALB WT AOE, n = 13 16 6.802396
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BALB WT AOE, n = 13 32 1.615039
BALB WT AOE, n = 13 32 12.56136
BALB WT AOE, n = 13 32 1.751621
BALB WT AOE, n = 13 32 10.4606
BALB WT AOE, n = 13 32 3.405692
BALB WT AOE, n = 13 32 3.046979
BALB WT AOE, n = 13 32 5.138819
BALB WT AOE, n = 13 32 6.355516
BALB WT AOE, n = 13 32 10.20448
BALB WT AOE, n = 13 32 6.64443
BALB WT AOE, n = 13 32 18.37788
BALB WT AOE, n = 13 32 3.286127
BALB WT AOE, n = 13 32 7.119519
BALB WT AOE, n = 13 64 2.26746
BALB WT AOE, n = 13 64 41.30844
BALB WT AOE, n = 13 64 3.642246
BALB WT AOE, n = 13 64 25.19357
BALB WT AOE, n = 13 64 3.559284
BALB WT AOE, n = 13 64 3.988721
BALB WT AOE, n = 13 64
BALB WT AOE, n = 13 64
BALB WT AOE, n = 13 64 16.89222
BALB WT AOE, n = 13 64 16.6267
BALB WT AOE, n = 13 64 27.31606
BALB WT AOE, n = 13 64 3.884046
BALB WT AOE, n = 13 64 10.5643
BALB WT AOE, n = 13 128 5.324903
BALB WT AOE, n = 13 128 87.22773
BALB WT AOE, n = 13 128 5.742002
BALB WT AOE, n = 13 128 42.09997
BALB WT AOE, n = 13 128 5.576558
BALB WT AOE, n = 13 128 5.516759
BALB WT AOE, n = 13 128
BALB WT AOE, n = 13 128
BALB WT AOE, n = 13 128 16.87386
BALB WT AOE, n = 13 128 25.99337
BALB WT AOE, n = 13 128
BALB WT AOE, n = 13 128 13.79561
BALB WT AOE, n = 13 128 17.65765
Muc5ac KO AOE, n = 4 4 1.99926
Muc5ac KO AOE, n = 4 4 1.402983
Muc5ac KO AOE, n = 4 4 2.209586
Muc5ac KO AOE, n = 4 4 1.410925
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Muc5ac KO AOE, n = 4 8 1.896724
Muc5ac KO AOE, n = 4 8 2.349068
Muc5ac KO AOE, n = 4 8 1.921075
Muc5ac KO AOE, n = 4 8 1.775477
Muc5ac KO AOE, n = 4 16 2.520983
Muc5ac KO AOE, n = 4 16 2.243595
Muc5ac KO AOE, n = 4 16 1.819613
Muc5ac KO AOE, n = 4 16 2.262319
Muc5ac KO AOE, n = 4 32 2.632477
Muc5ac KO AOE, n = 4 32 3.13292
Muc5ac KO AOE, n = 4 32 2.195089
Muc5ac KO AOE, n = 4 32 2.934794
Muc5ac KO AOE, n = 4 64 2.635094
Muc5ac KO AOE, n = 4 64 5.007348
Muc5ac KO AOE, n = 4 64 6.351069
Muc5ac KO AOE, n = 4 64 7.515857
Muc5ac KO AOE, n = 4 128 2.947929
Muc5ac KO AOE, n = 4 128 4.40801
Muc5ac KO AOE, n = 4 128 7.924337
Muc5ac KO AOE, n = 4 128 19.78577
BALB WT AOE + P-3001, n = 7 4 1.255069
BALB WT AOE + P-3001, n = 7 4 1.357685
BALB WT AOE + P-3001, n = 7 4 1.220027
BALB WT AOE + P-3001, n = 7 4 1.713947
BALB WT AOE + P-3001, n = 7 4 1.309743
BALB WT AOE + P-3001, n = 7 4 1.310526
BALB WT AOE + P-3001, n = 7 4 1.472925
BALB WT AOE + P-3001, n = 7 8 1.268291
BALB WT AOE + P-3001, n = 7 8 1.74836
BALB WT AOE + P-3001, n = 7 8 1.90285
BALB WT AOE + P-3001, n = 7 8 1.67477
BALB WT AOE + P-3001, n = 7 8 2.202705
BALB WT AOE + P-3001, n = 7 8 1.30699
BALB WT AOE + P-3001, n = 7 8 0.790453
BALB WT AOE + P-3001, n = 7 16 1.052877
BALB WT AOE + P-3001, n = 7 16 6.132445
BALB WT AOE + P-3001, n = 7 16 4.106292
BALB WT AOE + P-3001, n = 7 16 1.688387
BALB WT AOE + P-3001, n = 7 16 4.978462
BALB WT AOE + P-3001, n = 7 16 2.176566
BALB WT AOE + P-3001, n = 7 16 0.953341
BALB WT AOE + P-3001, n = 7 32 1.198506
BALB WT AOE + P-3001, n = 7 32 5.158922
85


BALB WTAOE + BALB WTAOE + BALB WTAOE + BALB WTAOE + BALB WTAOE + BALB WTAOE + BALB WTAOE + BALB WTAOE + BALB WTAOE + BALB WTAOE + BALB WTAOE + BALB WTAOE + BALB WTAOE + BALB WTAOE + BALB WTAOE + BALB WTAOE + BALB WTAOE + BALB WT AOE + BALB WTAOE +
P-3001, n = 7
P-3001, n = 7
P-3001, n = 7
P-3001, n = 7
P-3001, n = 7
P-3001, n = 7
P-3001, n = 7
P-3001, n = 7
P-3001, n = 7
P-3001, n = 7
P-3001, n = 7
P-3001, n = 7
P-3001, n = 7
P-3001, n = 7
P-3001, n = 7
P-3001, n = 7
P-3001, n = 7
P-3001, n = 7
P-3001, n = 7
32 5.678525
32
32 6.922513
32 2.497327
32 1.844692
64 1.31687
64
64 6.222619
64
64 10.61356
64 7.352855
64 5.777243
128 1.477769
128
128 11.59396
128
128 23.4412
128 10.07361
128 7.228725
86


APPENDIX C
R Code
setwd("~/A.Lab-Thesis.stuff/Mucolytic results")
## Unchallenged vs Challenged
# read in the data
first = read.csv("AHR.2.csv")
# prepare the data
head(first) #view firs few rows of data
sum data = ddply(first, c("Group", "Dose"), summarise,
N = sum(!is.na(Rrs)), mean = mean(Rrs, na.rm=TRUE), sd = sd(Rrs, na.rm=TRUE), se = sd / sqrt(N)) summary(first) plot(Rrs) plot(first$Rrs) lm(x ~ y)
# Use position dodge to move overlapped errorbars horizontally dev.new()
ggplot(sum_data, aes(x=Dose, y=mean, group=Group, color=Group)) + geom_errorbar(aes(ymin=mean-se, ymax=mean+se), width=.l, position=position_dodge(0.05)) + geom_line() + geom_point() +
scale_colour_manual(values = c("#DD035B","#67BB00")) +
labs(title="Airway Hyperreactivity",x= expression(paste("MCh ("*mu*"g/kg/min, iv)")),
87


y = expression(paste("Respiratory Resistance (", cmH[2]*0~"/ml/s"A"-l"*")"))) +
theme(plot.title = element_text(hjust = 0.5)) + scale_y_continuous(limits=c(0, 32.5))
### Unchallenged, challenged, and Muc5ac KO
# read in the data
sec = read.csv("AHR.3.csv") sec$Group = as.factor(sec$Group) sec$Dose = as.factor(sec$Dose) head(sec) #view firs few rows of data summary(sec)
sum_data2 = ddply(sec, c("Group", "Dose"), summarise,
N = sum(!is.na(Rrs)), mean = mean(Rrs, na.rm=TRUE), sd = sd(Rrs, na.rm=TRUE), se = sd / sqrt(N))
# Use position dodge to move overlapped errorbars horizontally dev.new()
ggplot(sum_data2, aes(x=Dose, y=mean, group=Group, color=Group)) + geom_errorbar(aes(ymin=mean-se, ymax=mean+se), width=.l, position=position_dodge(0.05)) + geom_line() + geom_point() +
scale_colour_manual(values = c("#DD035B", "chocolate 1", "#67BB00")) + labs(title="Airway Hyperreactivity",x= expression(paste("MCh ("*mu*"g/kg/min, iv)")), y = expression(paste("Airway Resistance (", cmH[2]*0~"/ml/s"A"-l"*")"))) + theme(plot.title = element_text(hjust = 0.5)) +
88


scale_y_continuous(limits=c(0, 32.5))
mm All four
# read in the data
Muc_mod = read.csv("Mucolytics.F+tc.csv")
Muc_mod$Group = as.factor(Muc_mod$Group)
Muc_mod$Dose = as.factor(Muc_mod$Dose) head(Muc_mod) #view firs few rows of data summary(Muc_mod)
sum_data3 = ddply(Muc_mod, c("Group", "Dose"), summarise,
N = sum(!is.na(Rrs)), mean = mean(Rrs, na.rm=TRUE), sd = sd(Rrs, na.rm=TRUE), se = sd / sqrt(N)) b oxplot(sum_data3) dev.new()
plot(Muc_mod$Dose, Muc_mod$Rrs, data(Muc_mod), colour = Muc_mod$Group)
# Use position dodge to move overlapped errorbars horizontally dev.new()
ggplot(sum_data3, aes(x=Dose, y=mean, group=Group, color=Group)) + geom_errorbar(aes(ymin=mean-se, ymax=mean+se), width=.l, position=position_dodge(0.05)) + geom_line() + geom_point() +
scale_colour_manual(values = c("#650EB0", "#DD035B", "chocolate 1","#67BB00")) + labs(title="Airway Hyperreactivity",x= expression(paste("MCh ("*mu*"g/kg/min, iv)")),
89


y = expression(paste("Airway Resistance (", cmH[2]*0~"/ml/s"A"-l"*")"))) + theme(plot.title = element_text(hjust = 0.5)) + seal e_y_conti nuous(limits=c(0,32.5))
####### Power Tests ##### challenged = first$Rrs sd(challenged, na.rm = TRUE)
power.t.test(n = 13, 0.2, sd = 10.62247 power = NULL)
###********************** *#####
#Perform ANOVA to get F value and P value, fitl = aov(Rrs ~ Group, data = Muc mod) summary(fit_l)
#Try some comparisons with the multicomp package, fitmc = glht(fit_l, linfet = mcp(Group = "Dunnett"), alternative = "less")
summary(fit_mc, test = adjusted(type = "single-step")) fit mc = glht(fit_l, linfet = mcp(Group = "Dunnett"), p.adjust.methods = "none") summary(fitmc)
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Full Text

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EFFICACY OF MUCOLYTIC TREATMENT IN DECREASING AIRWAY RESISTANCE AND INCREASING MUCUS CLEARANCE IN A MOUSE MODEL OF ASTHMA by LESLIE ELIZABETH MORGAN B.H.S., University of Missouri, 2012 A thesis submitted to the Facul ty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Master of Integrated Science s Integrated Sciences Program 2017

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ii This thesis for the Master of Integrated Science s degree by Leslie Elizabeth Morgan h as been approved for the Integrated Sciences Program b y Christopher Evans, Chair Marc Goalstone Loren Cobb May 13, 2017

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iii Morgan, Leslie Elizabeth (MIS, Integrated Sciences Program ) Efficacy of M ucolytic T reatment in D ecreasing Airway R esistance and I ncreasing M ucus C learance in a M ouse M odel of A sthma Thesis directed by Associate Professor Christopher M. Evans ABSTRACT Asthma is a serious disease that affects over 25 million people in the United States. Presently, treatment regimens for asthma have focused primarily on controlling smooth muscle contraction (bronchoconstriction) and inflammation. Another feature of asthma, mucus plugging, is prominent in fatal asthma. Mucus hypersecre tion is thought to contribute to mild to moderate disease, but its role in non fatal asthma exacerbations is less clear. This poor understanding of the role of mucus in asthma stems from a lack of knowledge about the production and function of mucin glyco p roteins. To this end, recent findings using sensitive molecular and genetic analyses have begun to clarify how mucus affects asthma. Specifically, the polymeric mucin glycoprotein MUC5AC is selectively overproduced in human asthma, and in mice it is requi r ed for airway hyperresponsiveness (AHR), a pathophysiological readout of the asth ma phenotype. The purpose of my studies was to determine whether a compound that disrupts mucin glycoprotein polymers can prevent AHR. To accomplish this, a mouse model of all ergic asthma was created using BALB/cJ wild type (WT) mice challenged with Aspergillus oryzae extract (AOE). Mice were ventilated, and respiratory system resistance (Rrs) was measured at baseline, and in response to methacholine (MCh) induced smooth muscle contraction and m ucin secretion. Rrs increased eight fold in AOE challenged WT mice compared to u nchallenged non allergic mice. Treatme nt of AOE challenged WT mice with the mucolytic compound P 3001 disrupted

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iv mucin polymers and prevented AHR. Histology sh owed that occlusion of the airways with mucus was improved in mucolytic treated WT mice In summary, by demonstrating that a novel mucolytic compound is capable of reversing AHR and mucus plugging in mice via disruption of mucin polymers and increased mobi lization of mucus, I have shown that reversing mucus hypersecretion is a potential therapeutic strategy for allergic asthma. The form and content of this abstract are approved. I recommend its publication. Approved: Christopher M. Evans

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v ACKNOWLEDGEMENTS The use of animals in this study was authorized under the University of Colorado Denver Institutional Animal Care and Use Committee ( IACUC ) protocol number: B 97414(12)1E This study was conducted with the assistance of the following grants: HL123442, NIH (Schwartz PI), 7/1/2014 6/30/19 I would first like to thank Dr. Martin Huber of the Master of Integrated Sciences Program for all of his support and encouragement throughout my time at University of Colorado Denver. I would also like to thank my thesis committee, Dr. Marc Goalstone and Dr. Loren Cobb. Profound thanks to the Chair of my co mmittee, Dr. Christopher Evans, who showed me consistent kindness, patience, support, and direction when I needed it. My eternal gratitude to you for affording me this amazing opportunity. Thanks also to Dr. Adrianne Stefanski for all of her sound advice and guidance with everything from bench work to professional etiquette. Thanks to all of my cowo rkers in the Evans lab for your assistance, comradery, and for putting up with all of my shenanigans. Naoko Vanessa, Rachel, Anna, and to Amanda for listening to all of my woes. Of specia l note, Dorota Raclawska for go to person for all things lab related people will forget what you did, but people will Maya Angelou

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vi DEDIC ATION I would like to express my profound gratitude to my husband Dan for providing me with unfailing support and continuous encouragement throughout my years of study and through the process of researching and writing this thesis. This accomplishment would not have been po ssible without you. A small nod to Oscar for doing what you do. Thank you. Leslie Morgan

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vii TABLE OF CONTENTS CHAPTER I INTRODUCTION ................................ ................................ ................................ .......... 1 Background and Review of Li terature ................................ ................................ ........... 1 Overview ................................ ................................ ................................ ............ 1 Innovation ................................ ................................ ................................ .......... 1 Impact of Asthma ................................ ................................ ................................ ........... 5 Pub lic Health Significance ................................ ................................ ................. 5 Current Therapeutic Strategies ................................ ................................ .......... 6 Pathoph ysiology of Asthma ................................ ................................ ........................... 7 Allergic Inflammation ................................ ................................ ........................ 7 Airflo w Obstruction in Asthma ................................ ................................ ......... 9 Airflow Obstruc tion: Contractile and Non Con tractile Components .............. 10 Smooth Muscle ................................ ................................ .................... 10 Edema ................................ ................................ ................................ .. 10 Mucus ................................ ................................ ................................ ... 10 Muc us Hypersecretion in Asthma ................................ ................................ ................ 11 Pathological Findings in Fatal Asthma ................................ ............................ 11 Sources of Mucus and Ontogeny o f Mucin Producing Cells .......................... 12 Production ................................ ................................ ................................ ........ 15 Secretion ................................ ................................ ................................ .......... 16 Mucolytic Therapy ................................ ................................ ........................... 17 Scientific Premise ................................ ................................ ................................ ........ 21

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viii Specific Aims ................................ ................................ ................................ ............... 21 II MUCOLYTICS IN A MOUSE MODEL OF ALLERGIC ASTHMA ............................. 23 Ra tionale ................................ ................................ ................................ ...................... 23 Research and Design Methods ................................ ................................ ..................... 23 Mice ................................ ................................ ................................ ................. 24 As thma Model ................................ ................................ ................................ .. 24 Surgical Preparation ................................ ................................ ......................... 26 Drug Administration ................................ ................................ ........................ 27 AHR Measurements ................................ ................................ ......................... 27 Histology ................................ ................................ ................................ .......... 28 Muc5b Protein Assays ................................ ................................ ..................... 30 Statistical Analysis ................................ ................................ ........................... 31 Results ................................ ................................ ................................ .......................... 32 AHR Measurements ................................ ................................ ......................... 32 Histology and Western Blots ................................ ................................ ........... 42 Discussion ................................ ................................ ................................ ........ 47 III. FUTURE DIRECTIONS ................................ ................................ .............................. 52 Microscopy Methods ................................ ................................ ................................ ... 52 Microscopy Results ................................ ................................ ................................ ...... 54 Carbon Nanoparticle Methods ................................ ................................ ..................... 57 Conclusions and Impact of Findings ................................ ................................ ............ 60 RE FERENCES ................................ ................................ ................................ ........................ 61

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ix APPENDIX A. Protocols for flexiVent and Western Blots ................................ ............................ 67 B. Flexivent Raw Data ................................ ................................ ................................ 8 1 C. R Code ................................ ................................ ................................ ................... 86

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1 CHAPTER I INTRODUCTION B ackground and Review of Literature Overview Asthma is characterized by airwa y inflammation, smooth muscle contraction, and mucus hypersecretion that collectively contribute to airflow obstruction. Airway hyperresponsiveness ( AHR ) is defined as an exaggerated degree of airflow obstruction in response to bronchoconstricting agents i n asthma patients in comparison to non diseased control subjects. Accordingly, compared to healthy individuals, people with asthma have a lower threshold to inhaled methacholine ( MCh ) (Bates et al. 2009), which is observed as potentiated airflow impairment in response to lower doses of MCh. AHR is present in asthma patients at baseline, and AHR correlates with asthma exacerbation magnitude and frequency. Innovation Current dogma emphasizes the significance of inflammation and smooth muscle contraction as the main causes of airflow obstruction in asthma (Fanta 2009). However, post mortem studies conducted on patients wh o died of acute asthma, showed marked mucus airway occlusion ( Fig. 1 ) (Evans and Koo 2009; Kuyper et al. 2003). Airflow obstruction can lead to air trapping causing lung hyperinflation ( Fig. 2 ). These features are evident in the lungs of a 51 year old fema le fatal asthma victim ( Fig. 3 ). Collectively, these pathological findings demonstrate both lung hyperinflation (the result of airflow obstruction) and mucus plugging. Mucus overproduction has been shown to occur in patients with even mild to moderate dise ase (Ordonez et al. 2001). Nonetheless, since mucolytics do not appear in the normal treatment regimen for asthma, their usefulness in asthma is poorly understood. In

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2 Figure 1 Mucus plugging in a fatal asthma attack Segmental bronchus in fatal adult onset asthma, showing oc clusive mucoid plugging ( arrow ) (Sidebotham and Roche 2003).

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3 Figure 2 Lung hyperinflation in fatal asthma. Right lung from fatal asthma in a young adult. The lung remained inflated after removal with sharp margins to the lobes. The imprints of the ribs and intercostal spaces produce d a corrugated effect on the lateral pleural surfaces of the lung (Sidebotham and Roche 2003).

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4 Figure 3 Mucus plugging and hyperinflation in a 50 year old fatal asthma victim. Lungs from a fatal asthma patient showing regional hyperinflation in the right upper lobe ( left, blue arrow ) compared to the left lower lobe ( left, black arrow ). A mucus plug occludin g a large airway ( right, arrow )

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5 part, this is due to an incomplete understanding of the chief macromolecular components of mucus, polymeric mucin glycoproteins. To this end, a recent study found that the mucin protein Muc5ac was required for AHR in mouse Although these studies suggest that protection in asthma could be gained by preventing mucus hypersecretion, it is not known if effective therapeutic efficacy can be achieved with mucol ytics, whose benefit is gained by reversing the effects of mucus after it is secreted. Accordingly, testing mucolytic agents may determine if they will inhibit the effects of Muc5ac in the airways of mice, and thereby provide a rationale for their developm ent as therapeutics in humans. In this vein, therapies could focus on using mucolytics to directly reduce airway occlusion by mucus, or they could be used as adjunct treatments to enhance the delivery and efficacy of mainstay drugs such as bronchodilators and glucocorticoids. Impact of Asthma Public Health Significance Asthma affects over 25 million people in the United States alone, including 7 million children (CDC: Asthma Fastats 2016). According to the Centers for Disease Control, asthma poses both health and economic concerns. In 2010, asthma costed approximately $56 billion, caused 10.5 million missed days of school, and resulted in 14.2 million missed days of work in the United States (CDC: Asthm a Fastats 2016). Missed school and work days can have profound impact s on entire families, especially if the family is economically disadvantaged Furthermore, the proportion of people with asthma has increased by 15% in the past decade. In aggregate, effe ctive treatments are needed to reduce the number of people affected by asthma and lessen the impact that it has on our society.

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6 Despite the staggering cost of asthma in the United States, it is important to note that not all people with asthma do not recei v e adequate treatment, or in some cases any treatment at all. Black and Puerto Rican children are more likely to have asthma than white children (CDC: Asthma Fastats 2016). Asthma is also more prevalent in persons that did not attain a high school diploma, and those living below the poverty level (CDC: Asthma Fastats 2016). Unfortunately, t here are dramatic differences in the care of people with asthma when considering these demographics. For instance, nearly one in four black adults cannot afford routine d octor visits and asthma medications. As a result, they are two to three times more likely to die from asthma than any other racial group (CDC: Asthma Fastats 2016). Developing new compounds that will be effective at dissolving mucus plugs will be instrumen tal at reducing asthma deaths, a major public health concern especially for people that fall into disadvantaged demographic groups. Current T herapeutic S trategies Most drugs used to treat asthma fall into one of two categories based on the following activities: (1) controlling inflammation or (2) controlling bronchospasm (Zdanowicz 2007). On the other hand, mucus is often ignored or undervalued in its role in asthma: Mucus was inflammation (Rogers 2004). In part, this is because the use of existing mucolytics has been shown to be largely ineffective (Aliyali et al. 2009), or even detrimental, with increased airflow obstruction noted via spirometry (Hirsch et al 1967). Because the acid dissociation constant (pK a ) of current mucolytics for breaking disulfide bonds is not advantageous, high concentration of drug is needed to be effective (Aliyali et al. 2010; Hays and Fahy 2003). Higher concentrations of drug would be more likely to elicit bronchospasm which is to be avoided in people with asthma As a result,

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7 mucolytics are rarely used in the treatment of asthma and treatment for mucus plugs in severe asthma is mostly supportive (Hays and Fahy 2003). Pathophysiology of Asthma Al lergic Inflammation Asthma can be characterized by inflammation and airflow obstruction. Inflammatory triggers include particulates, infectious agents, and allergens. Because allergic asthma is very common and is easily and reproducibly modeled in mice, my work focuses on studies in a mouse model of asthma that is driven by allergic inflammation. Allergic inflammation consists of an early and a late phase When a person with asthma inhales an allergen that they are sensitized to, the allergen is recognized by immunoglobulin E (IgE) which binds to mast cells, immediately triggering mast cell degranulation (Carroll et al. 2002). Mast cells release their contents, which include inflammatory mediators such as histamine, leukotrienes, prostaglandins and platelet activating factor (Rogers 2004). These mediators induce bronchospasm and can promote early phase inflammation. The late phase is characterized by eosinophilia and airway remodeling that is typified by mucous cell metaplasia, a process driven by the induct ion and overproduction of the mucin glycoprotein MUC5AC by conducting airway surface secretory cells (Caramor i et al. 2004; Evans et al. 2015 ; Ordonez et al 2001). Accordingly, post mortem exams of fatal status asthmaticus show pathologic features such as chronic inflammatory changes, eosinophilic infiltration, smooth muscle hypertrophy, and airway remodeling with mucus cell meta plasia ( Fig. 4 ) (Aikawa et al. 1992; Gronenberg et al. 2002; Sidebotham and Roche 2003 ).

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8 Figure 4. Post mortem pathological features in fatal asthma. Bronchiole in a fatal asthma patient indicat ing inflammation of the airway wall ( green arrow ) goblet cells in the epithelium ( red arrow ) and mucoid plugging of the lumen ( black arrow ) with intralumin al eosinophil leucocy tes (Sidebotham and Roche 2003)

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9 The validity of the present studies is reliant on the accuracy of our animal model for human asthma. Allergic asthma has been studied in numerous animal models including large animals (horses and sheep) and smaller animals including dogs, rabbits, guinea pigs, rats, and mice Each has advantages and disadvantages Large animals have the benefit of exhibiting spontaneous asthma but incur much higher costs to study Smaller animals allow for studies with h igher numbers, with greater control of their genetics (inbred and genetically engineered strains), and with much lower costs to the investigator. Mice have become an obvious and convenient animal model for asthma due to the plethora of tools and techniques available to study them along with a decreased cost and relatively short gestation period. A challenge with small animals is that they do not exhibit spontaneous asthma Rather, they are sensitized using a variety of triggers including chicken egg albumi n, house dust mite, cockroach, plant material, and fungi For this study, a mouse model of allergic asthma was developed by a series of challenges with aerosolized fungal allergen made from an Aspergillus oryzae extract (AOE). Once the challenges were complete, mice were studied during the late phase of inflammation 48 hours after the last AOE challenge Airflow Obstruction in Asthma In humans, r e ductions in airflow can be measured by a forced expiratory volume over one second (FEV 1 ) of less than 80% height. In acute asthma attacks, airflow obstruction can be reversible to some degree, as evidenced by an increase in FEV 1 2 adrenergic receptor agonist (Egan et al. 2009) Decreased expiratory flow rates are the result of airway lumen narrowing and occlusion in the conducting airways. Patients with asthma have been noted to have AHR, which is defined as increased sensitivity of the airways to

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10 bronchoconst ricting agents This narrowing can be due to smooth muscle mediated bronchoconstriction, or to non contractile obstructions, such as airway edema, inflammation, and excessive mucus in the airway, or to a combination of both contractile and non contractile factors. Airflow O bstruction in A sthma : C ontractile and N on C ontractile Components Smooth Muscle It is clear that the heightened sensitivity of asthmatic patients to MCh reflects contractions of the airway smooth muscle Once a patient has established air way inflammation and AHR with asthma, smooth muscle mediated bronchoconstriction can be triggered by a variety of endogenous factors as well. These include, but are not limited to: inhalation of irritants or cold air, exercise, emotional stress, hyperventi lation, and pharmacologic agents such as methacholine and histamine (Egan et al. 2010) In addition to this well recognized contractile component, non contractile factors also result in airflow obstructions. Edema Non contractile airflow obstruction can be caused by edema related to inflammation or by excessive mucus secreted into the airways Inflammation resulting from infection or allergen exposure can result in swelling of the tissues that surround bronchial airways, causing the airway walls to expand, thereby narrowing the passage of air Edema may be sensitive to glucocorticoid treatments, which are anti inflammatory and presumably reduce swelling. Mucus The other prominent, non contractile feature of airflow obstruction in people with asthma is excessive mucus in the airways. In non diseased states, mucus is protective and

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11 necessary for airway defense (Roy et al. 2014). The mucus layer coats the airway over a carpet of cilia that propel mucus and materials contained within mucus to the oropha rynx, where harmful foreign particles can be eliminated by expectoration or swallowing. This system is known as the mucociliary escalator (Egan et al. 2009; Knowles and Boucher 2002), and it appears to exist as a periciliary gel on brush structure (Button 2012) Changing the abundance or the viscosity of mucus can adversely affect these protective mechanisms ( Fahy and Dickey 2011; Sheehan et al. 1995; Yuan et al. 2015). Mucus Hypersecretion in Asthma Pathological F indings in F atal A sthma In individuals with chronic lung disease, the normal mechanisms of airway defense are disrupted, in part through overproduction of mucins. As a result of the increase in mucin production and secretion, the airway accumulates more mucus, which can diminish or occlude the airw ay lumen (James & Carroll 1995; James et al. 1989) When combined with the effects of smooth muscle mediated bronchoconstriction, this occlusion can dramatically potentiate airflow obstruction, or be fatal (Carroll et al. 2002; Sheehan et al. 1995). In aut opsied lung tissues from patients who die of fatal asthma episodes, airway occlusion by mucus is noted to be a ubiquitous feature ( see Fig s 1 and 2 ) (Gronenberg et al. 2002; Kuyper et al. 2003; Sheehan et al. 1995; Sidebotham and Roche 2003 ). Consequently there is a need for pharmacologic compounds that can control mucus in asthma and potentially treat patients once they have progressed to a state of mucus hypersecretion with mucus plugging. In fatal asthma attacks, the mucus of the victims was found to be abnormally solid in its composition (Sheehan et al. 1995). Studies conducted as early as the 1920s, when many diagnostic tools were not available, were able to make visual observations of this

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12 phenomenon post mortem (Houston et al. 1953; and Huber and K oessler 1922; James et al. 1989). With mucus plugging comes hyperinflation and air trapping as shown in Fig. 4. According to Sidebotham and Roche (2003), asthma is a preventable cause of mortality caused by a knowledge gap that persists despite our advance d understanding of the disease, and the pharmaceutics available for treatment, (Sidebotham and Roche 2003). These authors attributed these deaths to patients being misdiagnosed and undertreated (Sidebotham and Roche 2003). Indeed, despite the fact that al most all post mortem studies of fatal asthma described mucus airway occlusion as the cause of death, there are currently no efficacious mucolytics on the market. In order to research potential mucolytic compounds, we need to investigate these mucin protein s more closely at molecular and biochemical levels. Recent studies show that mucus secretions in fatal asthma are unusually tenacious, due to several different factors including the high concentrations of mucin proteins, their low charge density, and their extremely large size, their (Sheehan et al. 1995) Sources of M ucus and O ntogeny of M ucin P roducing C ells During healthy airway conditions, m ucus is comprised of mostly water (~ 98%) and is defined as, the free slime of mucous membranes, composed of glandular and surface cell secretions, along with various water inorganic salts, desquamated cells and leukocytes, DNA, and polypeptides ( Bansil and Turner 2002; Evans & Koo 2008; Sheehan et al. 1995). There are several types of mucin secretory cells in t he human airway. Of particular note are the goblet and club cells (formerly known as Clara cells). Goblet cells are more abundant in the upper airways, and virtually absent in the lower airways (Boers et al. 1999; Rogers 2003). Club cells are scant in the upper airways, and more prominent in the lower airways (Boers et

PAGE 23

13 al. 1999). Both of these mucin secreting cells have been shown to increase in number and size in states of chronic inflammation (Boers et al. 1999; Carroll et al. 2002; Dunnill et al. 1969; E van s et al. 2004, 2009; Rogers 2003 ). These increases can be problematic to the normal protective and clearance mechanisms of the human airway (Button et al. 2012). It has been shown that the number of club cells present in health is as follows : t he termi nal bronchioles, airway generation 15, contain about 11% club cells, and the respiratory bronchioles, airway generation 16, contain about 22% club cells (Boers et al. 1999). In allergic disease states such as asthma, there can be a dramatic increase in the amount of mucin producing cells in humans as well as mice (Evans et al. 2004 ; Ordonez et al. 2001 ). This increase results from allergic airway inflammation caus ing the production and storage of mucins, particularly MUC5AC/ Muc5ac (Evans et al. 2004 ; Ordone z et al. 2001 ). In aggregate, the increase in mucin production by secretory cells throughout the conducing airways results in increased numbers of mucin producing goblet cells, a phenotype that is referred to as to mucus cell metaplasia or hyperplasia Pa tients with asthma have increased of mucin secretory cells compared to healthy individuals ( Fig. 5 ). These changes can be very dramatic. Aikawa et al. (1992) show a 30 fold increase of goblet cells to total epithelial layer in three patients who had died o f status asthmaticus This same study These findings could contribute to the diminished capacity of the mucociliary escalator, leading to an increased amount of r etained mucus (Aikawa et al. 1992) Importantly, mucus hypersecretion also appears to be prominent in mild to moderate disease Ultimately, these factors lead to the release of excessive amounts of mucin glycoproteins into the airways in response to several triggers in asthma (Thornton and Sheehan 2004).

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14 Figure 5 Mucin secretory cell metaplasia. Photomicrographs of representative histologic sections from endobronchial biopsies from a healthy subject ( left ) and a subject with asthma ( right ). Mucin s tores in goblet cells appear as a mixture of blue and purple staining. The sections demonstrate increased Alcian blue and PAS staining in the secti on from the subject with asthma (Ordonez et al. 2001).

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15 Production Mucin glycoproteins are the predominant macromolecules in mucus and are responsible for its viscoelastic gel like properties (Bansil and Turner 2002) There are roughly nineteen mucin proteins encoded in the human genome The majority of these are cell tethered mucins ; the rest are s ecreted mucins (Thornton et al. 2008). Among the secreted forms, four polymeric/gel forming mucins have been shown to be expressed in the human airway MUC2, MUC5AC, MUC5B, and MUC19 (Evans & Koo 2009). Based on their expression levels and functional signi ficance in humans and mice, the two polymeric mucins that play the most prominent roles are MUC5B/Muc5b and MUC5AC/Muc5ac In mice, Muc5b is essential for the formation of a protective mucus gel (Roy et al. 2014), demonstrating that it is required for hom eostatic defense By contrast, Muc5ac is dispensable for baseline airway defense However, after allergen challenge, the airways can produce excessive levels of MUC5AC/Muc5ac (Evans et al. 2004; Thornton et al. 2008) In human airway epithelia, MUC5AC incr eases in the presence of chronic inflammation and is induced in asthma (Gronenberg et al. 2002; Ordonez et al. 2001). Further, Muc5ac was shown to be required for AHR in mouse models of asthma. In Muc5ac knockout ( Muc5ac / ) mice that had been challenged with aerosolized AOE for four consecutive weeks, the airways presented with marked inflammation, but failed to have significant AHR in response t o inhaled MCh (Evans et al. 2015 ). The data above suggest that MUC5AC would be an appropriate target for future pharmacologic compounds Inhaled corticosteroids have traditionally been used to combat airway inflammation, and they have also been shown to reduce MUC5AC expression and mucous meta plasia The anti mucous mechanism of action of corticosteroids is to mod ify the transcription of

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16 inflammatory mediators, thereby reducing inflammation, and potentially to also directly repress MUC5AC promoter activity and gene expression (Zdanowicz 2007) Indeed, glucocorticoid treatment has been shown to potently reduce MUC5A C gene expression in human lung epithelial cells in vitro (Chen et al. 2006, 2012, 2014; Takami et al. 2012; Hauber et al. 2007; Lu et al. 2005) However, in vivo studies of Muc5ac expression and mucous cell metaplasia in animal models have resulted in mix ed findings with steroid treatments Some studies show strong suppression of inflammation (Blyth et al. 1998; Lundgren et al. 2009 ) and others show mild (Yamabayashi et al. 2012; Bos et al. 2007) or no suppression (Mushaben et al. 2013; Kibe et al. 2003). It is not clear whether these conflicting results are due to species differences or the nature of experimental challenges Thus, while the anti inflammatory effects of corticosteroids are well established, their effects on mucin expression are variable and in many cases not efficacious once mucus secretion is already up regulated Hence at the very least these results reduce support for the utility of glucocorticoids as an effective strategy to reduce MUC5AC production and mucous cell metaplasia in asthma a direct anti mucus intervention is needed. Protein Assembly and Secretion Mucin production describes the manufacturing process of the mucin proteins in secretory cells. Secretion, on the other hand, describes the release of mucin prot eins on to the airway surface. There are several triggers for acute secretion such as, inflammatory mediators and neurotran smitters (Evans and Koo 2008). These triggers are activated by the AOE challenges and administration of MCh While mechanisms of sec retion are being studied by ot her groups ( Adler et al. 2013 ), i n this study, I investigated the efficacy of a mucolytic that acutely block s the effects of a post secretory event.

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17 Se creted mucin proteins are large (2 50 megadaltons), complex, highly glycosylated, protein polymers (Innes et al. 2009). A large portion of their mass is carbohydrates and these glycan chains form the large central domain of the mucin protein that absorbs copious amounts of water, leading to mucus with stro ngly viscous properties ( Fig. 6 ) (Thornton et al. 2008). Polymeric mucins, including MUC5AC/Muc5ac and MUC5B/Muc5b, have cysteine rich domains at their N and C termini, which are responsible for disulfide mediated gel forming properties (Thornton et al. 2008) These disulfide bonded termini can form long chains and branches of mucin oligomers (10s to 100s of microns long). Intramolecular disulfide bonds can also form in centrally located the cysteine rich domains. These bonds lead to the development of e xtremely high molecular weight polymers that have elastic properties Under healthy conditions the amounts of polymeric mucins and water are in equilibrium, creating a protective gel that is viscoelastic However, the presence of excessive mucin solids cau ses mucus to thicken dramatically, and can result in the formation of tenacious mucus plugs that are transported poorly Mucolytic Therapy Mucolytic T herapy Aerosolize d mucolytics could target the apoprotein backbones glycans or disulfide bonds An idea l mucolytic would thin mucus, making it is easier to expectorate. This would, in effect, aid the normal defense mechanisms of the airway and reduce mucus plugging. The use of a mucolytic could clear unwanted secretions from the airways which would increas e airflow This, in turn, could potentiate the effects and benefits of beta agonists and corticosteroids. A protease based mucolytic could be a useful pharmaceutical agent to disrupt the mucin polymer backbone. Research by Innes et al. (2009) showed that t he thick mucus

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18 Figure 6. Polymeric Mucin Proteins MUC5AC and MUC5B The apoprotien backbones of MUC5AC and MUC5B are comprised by an NH 2 termini (light grey) and COOH termini (dark gray). The central portion s are comprised of a mucin glycosylation domain (green). This region is rich in serines and threonines that are sites of O linked glycan attachments The glycosylation domain is interrupted by additional cysteine rich CysD domains (red). A : The COOH terminal von Willebrand like domain (VWD) is t he site of disulfide dimerization. Dimers can also assemble at the NH 2 termini via covalent disulfide bonds. m in length. B : Intramolecular d isulfide bonds can also assemble in the cystein e rich CysD s domain s. This occurs when hydropho bic amino acids form loops that may promote a mucin mesh network. C : In the mucin glycosylation domain, glycan linkages occur on the hydroxylated ends of serine and threonine residues through the initial attachment of N acetylgalactosamine (GalNac ). This i nitial O glycosylation step is followed by galactose and/or N acetylglucosamine (GlcNac) attachments, and can be elaborated further b y the attachm ent o f fucose, sial ic acid, and sulfate end groups (Evans et al. 2016 )

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19 present in acute asthma is laden with plasma proteins, which inhibit the degradation of mucins in a protease dependent manner (Innes et al. 2009). The polypeptide extensions that emerge from these domains are instrumental to the development of the mucin gel netwo rk, which leads to increased viscosity (Thornton et al. 2008). Another consideration might be to use a glycan inhibitor that would target the numerous glycosylated mucin domains. Current research into the glycans on mucin proteins is of current interest. There is some evidence for selective glycosylation of airway mucins in asthma, but these are poorly characterized and not adequately targeted. Disrupting mucus polymers by targeting disulfide bonds could loosen dense mucus aggregates and thereby restore he althy mucus functions ( Fig. 7 ). Indeed, at present, the best characterized, potentially safest, and most efficacious options are agents that work by breaking up the disulfide bonds of mucins (reducing agents). One such mucolytic currently on the market in the United States is N acetylcysteine (NAC, brand name Mucomyst ). NAC works by breaking up the disulfide bonds of mucus into weaker sulfhydryl bonds (Egan et al. 2010; Gardenhire and Rau 2008). This decreases the viscosity of the mucus, so it can be expec torated. N acetylcysteine has a short half life on airway surfaces, and it is a weak reducing agent at physiologic pH. For any therapeutic benefit to be seen, NAC must be given as an aerosol at a 1 M concentration, which has the adverse effect of irritatin g airways and triggering bronchospasm. Because of its potential as an effective therapeutic agent, our collaborators at Parion Sciences, as well as colleagues in other academic laboratories (Yuan et al. 2015), have placed effort into developing mucolytic agents that disrupt disulfide bonds with greater potency. My research plan seeks to determine whether delivering the phosphine compound P 3001, a fast

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20 Figure 7. Mucolytic action on disulfide bonds Disulfide bond s shown as both end to end formations (s s) at the N and C termini and as intramolecular formations in the cysteine rich domains ( s s ), contributing to thick and tenacious mucus ( left ). Application of P 3001, a phosphine based reducing agent could target and dis solve these bonds ( middle ), making mucus less viscous and easier to expectorate ( right ) (modified from Yuan et al. 2015)

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21 acting mucolytic that is at least 500 fold more potent than NAC, disrupts the mucin polymer network, thus suggesting that inhaled mucolytic therapy is an effective potential pharmacologic approach in the treatment of asthma and potentially other obstructive dis eases Scientific Premise Asthma is a serious and preventable disease. Despite our advanced understanding of the pathophysiology of asthma, and the pharmaceutics available for its treatment, its prevalence remains high, and patients still d ie from this d isease (Sidebotham and Roche 2003). Despite the fact that almost all post mortem studies of fatal asthma described mucus airway occlusion as the cause of death ( see Fig. 1), mucolytics are not being added to asthma treatment regimens This is due in part t o the limited availability of agents that are efficacious at tolerable concentrations (Gronenberg et al. 2002). This study seeks to determine whether a mucolytic agent with a similar mechanism of action to NAC, but with greater potency at breaking disulfid e bonds, could provide support for the efficacy of disrupting mucin polymer to improve airflow in an allergic asthma model. Specific Aims The goal of this study was to create a mouse model of asthma that would be useful for determining whether a pharmacolo gic agent is effective in treating mucus secretion and to test the effects of mucolytic treatment on AHR Aim 1. Create an allergic mouse model with mucus dependent AHR that will permit test ing of the effects of mucolytic intervention I adapted an existing protocol for studying AHR in AOE challenged WT mice This allowed the simultaneous testing of a bronchoprovocative agent, mucin secretagogue, and mucolytic intervention treatments.

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22 A im 2. Determine the effects of mucolytic intervention on AH R. Employing the model established above Aim 1, the effect of an inhaled reducing gent P 3001 on AHR was tested. Aim 3 Determine the effects of mucolytic treatment on airway mucus plugging and mucin polymer structure Histologic data was used to quantify the extent of inflammation and mucus hypersecretion present in the mouse models. Western blots were done to determine the extent of mucin protein breakdown in bronchoalveolar lavage samples post delivery of P 3001 or sa line

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23 CHAPTER II MUCOLYTICS IN A MOUSE MODEL OF ALLERGIC ASTHMA Rationale Mucus is a defining feature of asthma in humans (Gronenberg et al. 2002 ) ; and mucus plugging is a characteristic of fatal asthma attacks (Kuyper et al. 2003). In mice, M uc5ac is essential to allergic asthma like obstruction since Muc5ac / mice have previously been shown to be protected against AHR to inhaled MCh (Evans et al. 2015). The long term goal for my research is to determine whether an inhaled mucolytic could ultimately be beneficial for patients with asthma. This study is the first to deploy a mucolytic intervention in a mouse model of allergic asthma The experiments presented here were designed to establish a suitable model (Aim 1), demonstrate the efficacy of a test compound (Aim 2), and verify that the compound affected its target (Aim 3 ) Accordingly, the studies below test the central hypothesis that a fast acting mucolytic agent will prevent AHR by acut ely disrupting the formation of mucus plugs. A mouse model of allergic asthma was developed by a series of challenges with aerosolized AOE. Once the challenges were complete, mice received increasing dosages of intravenous MCh in the presence of aerosol a dministration of either saline or the mucolytic P 3001. Lung mechanics were measured in mechanically ventilated mice Total respiratory system resistance (R rs ) was calculated, and differences in R rs were determined between allergically inflamed and MCh tre ated mice that received P 3001 and those that did not. In addition, histology and Western blots were done to quantify the amount of mucus present in the airways, and to demonstrate the breakdown of mucin proteins among the two test groups.

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24 Research and De sign Methods Mice All studies were conducted with the approval of the University of Colorado Denver Institutional Animal Care and Use Committee (IACUC) For studies in wild type (WT) animals, BALB/cJ mice were used, as this strain is well studied in model ing allergic asthma In addition, Muc5ac / mice that were crossed onto a congenic BALB/cJ strain background were used to generate data for comparison to previous studies (Evans et al. 2015) Both male and female mice were used beginning at 6 8 weeks of a ge Mice were housed in specific pathogen free conditions in ventilated cages with no more than five mice per cage Mice were kept on a 12 hour light and dark cycle with the light cycle being 0600 1800. All mice were fed irradiated normal mouse chow and ha d access to food and water ad libitum Asthma M odel To model asthma like inflammation and mucous cell metaplasia, mice were challenged using aerosolized Aspergillus oryzae extract (AOE ; Sigma, catalog # P6110 ) AOE is a protease complex produced by submerged fermentation of A. oryzae. AOE has been shown previously to elicit an allergic inflammation in mice (Kheradmand et al. 2002; Evans et al. 2015) Aerosolized AOE challenges were given using an Ultravent jet nebuli zer Mice were loaded into custom built nose only exposure chambers, and a challenge aerosol was generated using 5 ml of a 10% vol/vol mixture of AOE and saline Challenges lasted until the full volume was delivered (approximately 40 45 minutes) (Evans et al. 2015). Mice received 4 weekly challenges, and endpoint analyses were studied 48 h after the last AOE challenge which corresponds with a late phase asthmatic response ( Fig. 8 ) Mouse challenges provide our model with inflammation, mucous cell metaplasia with associated mucus hypersecretion, and AHR

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25 Figure 8 Immunization and Aerosol Challenge protocol for Inducing Allergic Phenotypes and the flexiVent B ALB/cJ mice were challenged weekly with AO E. Measurements were then determined 48 hours after the last challenge ( top ). The Flex iVent small animal ventilator ( bottom ) (Scireq, Montreal Quebec, Canada).

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26 Surgical P reparation The techniques and measurements in my model of asthma present a challenge due to the small size of the animal Whereas human asthma is easily tested using non invasive procedures, noninvasive methods of attaining lung mechanics data in mice are not as accurate as invasive ones. Invasiveness does dictate that mice are s tudied under conditions that are far from a natural state (Bates & Irvin 2003), but the dichotomy between measurement precision and maintaining natural conditions was thoughtfully considered in the design of these studies. In this instance, experimental co ntrol and measurement precision was chosen over noninvasiveness. Assessments were made with the flexiVent (Scireq, Montreal Quebec, Canada), a ventilator used for measuring changes in respiratory system resistance (Rrs) and responses to inhaled and intrave nous ( IV ) drugs ( see Fig. 8 ) Th ese experiments were conducted in anesthetized, tracheostomized, paralyzed mice using the single frequency forced oscillation technique (SFOT), in which a sinusoidal oscillatory flow signal is applied to the airway opening w hile the airway pressure is measured (Bates et al. 2009). Mice were anesthetized with a 2 g/kg (0.01 ml/g) intraperitoneal (IP) dose of urethane Urethane provides deep sedation that has been shown previously to have a low impact on cardiac and respirator y sy stems ( Soma 1983). Furthermore, urethane induced anesthesia lasts for up to 2 h ou rs, although each experiment lasted approximately 20 minutes (Hara and Harris 2002) Once anesthetized, surgery was performed to cannulate the trachea with a beveled 18 ga uge blunt tip catheter Mice were then placed on the flex i V ent. Ventilated mice were paralyzed with an initial IP injection of 0.2 ml of a 20 mg/ml solution of succinylcholine chloride to prevent spontaneous respirations from interfering with Rrs

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27 measureme nts. In order to maintain paralysis throughout the experiment, the initial bolus was followed by a continuous infusion of succinylcholine administered IV Drug A dministration Once mice were paralyzed and stably ventilated, their abdomens were surgically opened for catheterization of the inferior vena cava (IVC), followed by a continuous infusion Continuous IVC infusion also contain ed a and succinylcholine chloride MCh is a synthetic cholinergic that stimulates muscarinic receptors to cause smooth muscle contraction and mucin secretion. MCh challenges for AHR studies can be administered as an aerosol, or by IV routes In order to rule out potential competition between MCh and mucolytic agents if they were aerosolized simultaneously, administration routes have been separated, and MCh was d elivered IV while mucolytic treatment was delivered by aerosol. Mice were then given either aerosolized sterile normal saline, or aerosolized mucolytic diluted in saline. Aerosols on the flexiVent were administered with an in line ultrasonic nebulizer (see below). AHR M easurements After placement on the flexiVent, 2 3 deep inflation maneuvers were performed to facilitate alveolar recruitment and normalize lung volumes ( Fig. 9 ) (Hantos et al 2003). Baseline values of respiratory mechanics were then assessed with several perturbations using 2.5 Hz single frequency forced oscillation techniques (SFOT) Each cycle included a default ventilation pattern consisting of: respiratory rate = 150/min, tidal volume = 10 ml/kg, pressure limit = 30 cmH 2 O PEEP = 3 cmH 2 O I nspiratory time = 0.16 s, and E xpiratory time = 0.2 s. In addition to the default ventilation pattern that ran the length of the experiment,

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28 several perturbations were executed per an automation script. These included, in order, two deep inflations, admi to determine Rrs for each of the 6 doses of MCh. Rrs is calculated in the flexiVent software by fitting the equation of motion of the linear single compartment model of lung mechanics to a SFOT data using multiple linear regressions (Shalaby et al. 2010). Since asthma is characterized by abnormal resistance to airflow in the lungs, as well as exaggerated responsiveness to agents that acutely cause obstruction, changes in Rrs were measu red in response to increasing doses of MCh (Mcgovern et al. 2013). AHR was defined as exaggerated Rrs responses to given doses of MCh between animals. To determine this, differences in Rrs changes in response to MCh were analyzed by performing linear regre ssion on log transformed dose response curves and testing for differences in the slopes of the regression lines for each mouse in each of the treatment groups. After the last dose of MCh was delivered, mice were removed from the ventilator and euthanized b y exsanguination. Mice were subsequently subjected to fixation of the lungs with a methanol based fixative consisting of: 60% methanol (100%), 30% chloroform, and 10% glacial acetic acid, for histology, or a bronchoalveolar lavage (BAL) for Western blots. Histology Fixed lungs were excised after 30 60 minutes and placed in a scintillation vial filled with methacarn. After 24 hours, lungs were removed and placed in a scintillation vial with 100% methanol. Methanol solutions were replaced with fresh solution again in 24 hours. After fixation in methanol, left lungs were cut into ~2 mm transverse sections and imaged. Lung volume was calculated using the Cavalieri method, or Archimedes principle (Krefft et al. 2015)

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29 Figure 9. FlexiVent script perturbations with dosages of MCh and P 3001. Mice received a default ventilation pattern of 150 breat hs/min. After placement on the flexivent, and prior to aerosol delivery, two deep inflations were administered ( top left ). A snap shot measurement, denoted by the sinusoidal waveform ( top right ), from which respiratory system resistance (Rrs) is derived. This cycle of Rrs measurements is repeated 20 times for each dose of methacholine (MCh). Dosages of MCh and P 3001 shown respectively ( bottom ).

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30 were collected on positively charged glass microscope slides. Tissues were stained with alcian bl ue periodic acid PAS) stain and examined with an Olympus BX63 microscope in order to establish degrees of mucin production and secretion. This was done to determine if mice treated with the mucolytic P 3001 showed more patent airways, compared to mice treated with only aerosolized saline Muc5b P rotein A ssays Western blots were performed on BAL specimens to determine the changes in mucin polymer sizes of specimens from mice that received P 3001 versus those that received only aerosolized sal ine. This was done to determine if P 3001 was effective at breaking down the mucin glycoproteins in the bronchoalveolar fluid (BALF) obtained after the flexivent experiments. BALF samples were first thawed in an ice/water slurry. A protease inhibitor was a dded to each sample (10% per sample volume). BALF for each sample and double deionized water were added to 1.5 ml tubes in order to of protein per lane, based on analysis from the dot blot loading buffer was added to each tube, and samples were loaded into the wells of a prepared agarose gel. The loaded agarose gel was covered with a running buffer, consisting of 1x Tris Acetate + ethylenediaminetetraacetic acid ( EDTA ) (TAE) + 0.1% so dium dodecyl sulfate (SDS), and allowed to run overnight in a 4 C cooler at 42 V. On day two, the agarose gel was placed in a transfer apparatus with a PVDF membrane and covered with saline sodium citrate (SSC). The apparatus w as attached to a vacuum pressure of 25 to 37.5 cm H 2 O for 90 minutes. Membrane was then removed and rinsed 2 3 times in 4x SSC and blocked with Odyssey buffer for one hour on a rocker.

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31 Primary antibody (rabbit anti mouse Muc5b ; 1:5000 dilution ) was app lied to membrane and cont ainer incubated overnight in 4C refrigerator with gentle rocking. On day three, the primary antibody was discarded and membrane was washed 3 4 times for 5 minutes each wash with 1x tris buffered saline + 0.05% Tween 20 (TBS T). Membrane was then probed with a secondary antibody (IRDye 680 RD Goat anti Rabbit; Lot# C41029 03; stored undiluted at 4C; used at 1:15,000 dilution) for one hour with gentle rocking Membranes were protected from light with a foil covered receptacle during this phase. Washings w ith TBS T were repeated as above and immunoreactivity was imaged in an Odyssey FC system (LI COR Lincoln, NE ). Statistical A nalysis Statistical analysis and graphs were performed in GraphPad Prism 7 (San Diego, CA) and R statistical software. Data from d ose response curves w ere log transformed extreme outliers were removed, and linear regression was performed. Data from mice that met the exclusion criteria below were also removed. The slopes for each group were compared using a o ne way ANOVA with Dunnett There were a few cases in which data were excluded. Criteria for exclusion of AHR data in mice consisted of ( 1) death of the animal prior to the test ( n = 5 ), ( 2) abnormally high baseline values not responsive to basic manipu lation, such as repositioning of the animal, the platform, or the cannula ( n = 3), ( 3) abnormally low baseline values, denoting a leak in the system with either the equipment or the animal, or incomplete paralysis resulting in spontaneous respiratory effor t, that could not be readily identified and corrected with either manipulation or bolus of paralytic agent ( n = 2), and ( 4) values that decreased and then increased again with the dose response curve (complete exclusion n = 11 ) For this last case,

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32 mice were not always completely excluded from the study Rather, their Rrs values that increased in a dose dependent manner were included, and all subsequent values ignored under the assumption that the cause of the decrease in values was due to death of the an imal This was observed in allergically inflamed mice, and the loss of dose responsiveness occurred after responses that were strong enough to kill the animal (evidenced by severe bradycardia that does not recover) Results AHR M easurements In Aim 1, I s ought to determine whether IV MCh challenge would be useful in my study design In response to IV MCh, AOE challenged WT mice showed higher Rrs values compared to unchallenged mice. The dose response curves showed a mean difference of approximately 8 fold in Rrs values for the AOE challenged mice compared to the unchallenged, non aller gic healthy control mice ( Fig. 10 ) Thus, AOE mice had a potentiated increases in resistance compared to that of the unchallenged WT mouse. This confirms that the AOE challeng ed WT mice exhibited increased AH R Previous studies have suggested that IV infusion of MCh induces solely airway smooth muscle constriction, as opposed to what is observed with aerosolized administration, which can trigger smooth muscle constriction and epithelial mucin secretion in the airways (Petak et al. 1997) I thus next determine d the role of mucin secretion in AHR as a response to IV MCh. It has been shown that the mucin protein Muc5ac is required for inhaled MCh in duced AHR in AOE challenged WT mice (Evans et al. 201 5 ). Therefore, I tested genetically deficient Muc5ac ( Muc5ac / ) mice to examine whether mucin dependent AHR to IV MCh was also present

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33 Figure 10 Ad ministration of AOE creates a suitab le model of allergic asthma and AHR in mice. Airway resistance results in BALB/cJ WT mice showing an approximate eight fold increase in respiratory system resistance (Rrs) of the AOE challenged WT mice compared to that of the unchallenged mic e after AOE ch allenges.

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34 I show ed that Muc5ac / mice demonstrated lower changes in Rrs in response to MCh than that of AOE challenged, untreated WT mice in response to IV administration of methacholine ( Fig. 11 ). The AOE challenged, non mucolytic treated WT mice showed higher changes in Rrs compared to AOE challenged Muc5ac / mice. Rrs of the AOE challenged WT mice is approximately three fold higher than that of the AOE challenged BALB Muc5ac / mice. This supports the previous finding that Muc5ac is required for AHR Th is difference between the dose response curves of the AOE challenged, non mucolytic treated WT mice and the AOE challenged Muc5ac / mice represents the mucus dependent component of AHR. The difference between the dose response curves of the AOE chal lenged Muc5ac / mice and the u nchallenged mice, represents the non mucus dependent component of AHR. The most likely contributors to the non mucus dependent component being smooth muscle mediated bronchoconstriction and inflammation. In aggregate, these studies in AOE challenged, Muc5ac / mice represent genetic prevention of AHR in response to IV MCh. Importantly, th ese also demonstrates that the IV MCh challenge protocol is well suited for analysis of topical t reatment with a mucoly tic agent, in that I was able to elicit the mucus dependent component of AHR with IV MCh. Having established the applicability of this model, I next sought to test the effects of mucolytic treatment in acute reversal of AHR. AOE challenged WT mice treated with aerosolized P 3001 showed substantially lower changes in Rrs in response to IV MCh comp ared to their AOE challenged, non mucolytic treated littermates and almost identical dose response curves to that the of AOE challenged Muc5ac / mice ( Fig. 12 ). The Rrs values of my mucolytic treated and unchallenged groups were similar, suggesting that there was a treatment effect in the P 3001

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35 Figure 11 Muc5ac is required for AHR in an AOE induced allergic mouse model of asthma. Airway resistance of BALB Muc5ac / mice in response to IV MCh challenges. Airway resistance of challenged WT mice is approximately three fold higher than that of the knockout mouse. Unchallenged and BALB WT AOE data a re the same as shown in Fig. 10 The difference between the dose response curves of the AOE challenged Muc5ac KO mice, and the AOE challenged WT mice represents the mucus dependent component of AHR. The difference between the unchallenged mice and the Muc5ac KO mice illustrates the non mucu s dependent component of AHR.

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36 Figure 12 P 3001 protects against AOE induced AHR in my allergic model of asthma. Airway resistance of AOE challenged mucolytic treated WT mice in response to IV MCh challenges. Airway resistance of AOE challenged untreated mice is approximately three times greater than that of the AOE challenged P 3001 treated mice. Unchallenged, BALB WT AOE, and BALB Muc5ac / data are the same as shown in Fig. 10 and 11

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37 treated group. AOE challenged untreated mice had Rrs measurements that were approximately two and a half times greater than that of the AOE challenged P 3001 treated WT mice demonstrating that AHR was effectively reduced with the application of the mucolytic P 30 01. The Rrs data was log (x) transformed outliers were removed, and linear regression was performed on the individual mice. The means of the slopes for each group in response to IV MCh are shown ( Fig. 13 ). The mean slope of the AOE challenged, non mucolyt ic treated WT mice was higher than that of the unchallenged mice 13.1 2.8 versus 1. 3 0. 3 respectively (data are shown SEM) The mean slope s of the AOE challenged Muc5ac / mice (demonstrating genetic prevention of AHR) and the AOE challenged P 3001 treated group (demonstrating acute reversal of AHR) were 3.4 0. 6 and 2. 9 0. 9 respectively They were both substantially lower that the AOE challenged, non mucolytic treated WT mice Individual regression slopes for each mouse after linear regre ssion of the log (x) transformed data is shown separated by group ( Fig. 14 ). A list of data from all mice used in this study with their respective individual slopes and r 2 values ( Table 1 ) A one way ANOVA was performed on the individual slope values from table 1 and compared with ( Fig. 15 ) The figure s are shown with mean SEM bars. The AOE challenged group was found to be statistically higher than all ot her groups of mice in this study (p value < 0.05). In summary, there w ere significant differences (p value < 0.05) in AHR between the AOE challenged P 3001 treated WT mice versus the AOE challenged, non mucolytic treated WT mice with significant p value illustrated by an asterisk ( see fig ure 15 ). The AOE challenged, P 3001 treated WT mice h ad almost identical slopes when compared to the AOE

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38 Figure 13 Summary of AHR in allergic mice. Above are the slopes after log(x) transformation by group.

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39 Figure 14 Individual regression slopes of log (x) transformed data by group Individual slopes for unchallenged mice shown in green ( n = 7, top left ). Individual slopes for AOE challenged, non mucolytic treated WT mice shown in magenta ( n = 12, top right ). Slopes for AOE challenged Muc5ac KO mice shown in orange ( n = 4, bottom left ). Slopes for the AOE challenged P 3001 treated WT mice shown in blue ( n = 7, bottom right ).

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40 Table 1. Individual slopes and R 2 values per group. Unchallenged Slope R squared 505 2. 8 0.8 1 506 0. 7 0.93 509 1.5 0.9 2 516 0.2 0.99 522 0.1 0.1 5 529 1.7 0.97 530 2. 2 0.9 3 AOE 514 29. 2 0.70 549 6.9 1 552 26.2 0.8 7 554 4.1 0.98 555 16. 6 0.8 7 584 4. 5 0.92 586 4. 4 0.6 6 597 10.8 0.95 606 16.1 0.95 607 22.0 0.97 613 6. 9 0.65 617 9. 9 0.90 AOE + P 3001 556 0.1 0.2 9 585 1. 3 1 .00 598 4. 6 0.96 614 0.0 0.41 615 7. 8 0.96 616 5.9 0.81 625 4.4 0.78 Muc5ac KO AOE 555 0. 0 0.86 556 2. 3 0.8 6 558 4.0 0.7 1 560 4.4 0.72

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41 Figure 15. AOE challenged, non mucolytic treated WT mice were significantly different than all other groups. One performed on individual slopes for each group showed that all groups were significantly (p value < 0.05, denoted by *) different from the allergically challenged, non mucolytic treated WT mice.

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42 challenged, Muc5ac / mice, which have previously been shown to be protected against AHR in response to AOE challenges and MCh administration. Histology and Western Blots Airways of a healthy, non AOE challenged BALB WT mouse tended to show pat ent airways free of mucus plugs. ( Fig. 1 6 ). This image shows a bronchus (a large central airway) that is completely free of mucus. The airways in these mice also appeared to have no airway thickening. T he AOE challenged non mucolytic treated BALB WT m ic e were prone to have mucus plugs that often occlud ed the larger airway s ( Fig. 1 7 ) This image shows a bronchus that almost totally occluded by a mucus plug with only a small airspace left. A situation such as this would prevent air from reaching all airways distal to this occurrence. This could be potentially an entire lobe of the lung. In regards to the AOE challenged BALB Muc5ac / mic e, airways tended to display some degree of mucus secretory cell metaplasia but airways usually remain ed patent ( Fig. 1 8 ). Some remaining mucus (Muc5b) is present in this image but it appears to remain in the secretory cells. The microscopic appearance of an AOE Challenged, P 3001 treated WT mice usually showed patent airways but there was often some muc o us cell metaplasia ( Fig. 19 ). As with the BALB Muc5ac / mouse, the mucus appears to remain confined to the secretory cells. This may be the result of mucociliary clearance of secreted mucus with the remaining signal resulting from residual intracellular mucin present in the treated mice. To confirm that P 3001 affected the mucin targets as predicted, Western blotting was performed under non reducing conditions With the Muc5b Western blot, the mucin polymers have been effectively br oken down (low molecular weight) in the P 3001 treated

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43 Figure 16 The patent airway of an unchallenged WT mouse Airways in an un challenged mouse display the absence of airway thickening and excessive mucus ( arrow ).

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44 Figure 1 7 Non mucolytic treated AOE challenged WT mouse with mucus airway obstruction Airways show mucus in a bronchus ( black arrow ) and marked mucus airway occlusion in the bronchiole ( yellow arrow ).

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45 Figure 1 8 A bsence of mucus plugging in a Muc5ac / mouse Histology showing the airway of an AOE challenged BALB Muc5ac / mouse. Note the airway thickening in the absence of mucus plugging ( arrow ).

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46 Figure 1 9 Absence of mucus plugs in a P 3001 treated WT mouse. AOE challenged mucolytic treated mouse showing airway thickening with the absence of mucus plugging ( arrow ).

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47 WT mice (Fig. 20). In contrast, the mucin proteins of the AOE challenged, non mucolytic treated WT mice have a higher molecular weig ht, indicating that the polymers are still intact. Discussion Asthma is a serious and sometimes fatal disease that affects 25 million people in the United States alone. Asthma is characterized by airway inflammation, smooth muscle contraction, and mucus hy persecretion that contribute to AHR. The current strategies in the treatment of asthma target the control of inflammation and smooth muscle contraction, crediting them as being the foremost causes of airflow obstruction. However, even though mucus hypersec retion and mucus plugging have been noted to be key features of mild to moderate disease and to fatal asthma attacks, these components are poorly resolved. A lack of therapeutic options stems from an incomplete understanding of the mechanisms of mucin medi ated obstruction. Under normal conditions, mucin glycoproteins are important for airway defense. However, in diseased states, mucus hypersecretion can be life threatening (Thornton et al. 2008). Mucin proteins are large and complex structures that should ideally be studied in vivo but there are few available models (Thornton et al. 2008). Consequently, research on novel pharmacologic treatments for mucus remains deficient but nascent. Recent literature has identified a specific polymeric mucin protein (Muc 5ac) as being responsible for airway hyperreactivity in mice (Evans et al. 2015). Although mucus plugging features prominently as the cause of death, mucolytics are not currently being added to treatment regimens. As previously noted, this is d ue, in part, to the lack of options available to clinicians. Dornase alfa, a mucolytic currently on the market, works by bre aking up the

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48 Figure 20 Mucin Protein breakdown in P 3001 treated WT mice Western blot on bronchoalveolar lavage fluid (BALF) samples from saline and P 3001 treated WT mice Results showed mucin glycoprotein breakdown in the mice that were treated with the mucolytic P 3001.

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49 extracellular DNA but not the mucin glycoproteins in a Rau 2008). This is particularly effective for patients with cystic fibrosis, where mucus is typically purulent (Gardenhire and Rau 2008). However, in cases of moderate to severe asthma Dornase alfa is not an effective mucol ytic, most likely because the relative amount of DNA in asthmatic compared to CF mucus is too low for the drug to be effective (Boogaard et al. 2008). Di stinct target s for mucolysis that target mucin glycoproteins are the cysteine rich domains that are re sponsible for disulfide bond mediated polymer formation. One of these N acetylecysteine (NAC, Mucomyst), is available on the market. However, it is very inefficient and must be given to patients at very high concentrations. A ccordingly, a n unfortunate sid e effect of NAC is bronchospasm, which is to be avoided in people with asthma since bronchospasm is a manifestation of asthma Future mucolytic compounds need to address the presence and viscosity of mucus, with higher efficacy at lower concentrations tha t do not cause airway contractilit y. Furthermore, new mucolytic drugs need to preserve Muc5b, as diminished amount of this mucin can result in increased mortality from respiratory infections (Roy et al. 2013) Having identified a target in airway mucus (M uc5ac), it is reasonable to assume that research can proceed to develop a novel pharmacologic compound to treat mucus occlusion in asthma, and potential ly decrease asthma fatalities. As noted above, hypersecretion of Muc5ac can be problematic, whereas the production of Muc5b is necessary Thus, f uture pharmacologic compounds need to diminish Muc5ac while preserving Muc5b. Additionally, asthma treatments should avoid any potential adverse effects such as bronchospasm or rapid liquification of airway secretions, as these events can occlude the airway.

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50 Although contractile forces are a contributing cause to airflow obstruction in asthma (An et al. 2007), contracti le forces alone are not adequate to explain the total airway occlusion noted in fatal asthma attacks. Mucus plugging features prominently in fatal cases of asthma (see 1, 2, and 3). I n a study by Kuyper et al. (2003) 275 airways from 93 cases of fat al asthma were examined. They found that mucus occlusion ranged from 4% to 100%, but only 5 cases showed less than 20% occlusion (Kuyper et al. 2003). This indicates that further research into the use of mucolytics in the treatment of asthma is appropriate My work illustrates the efficacy of a phosphine based mucolytic compound in the treatment of a mouse model of allergic asthma by reducing the viscoelasticity of mucus. My stud y employed techniques in mice that elicit ed an allergic response using an inhaled allergen and MCh delivery. I adapted existing methods in the laboratory by employing IV administration of MCh (the majority of previous studies have delivered MCh by aerosol). MCh was given IV in order to separate its de livery routes from the therapeutic mucolytic test compound P 3001, so as to prevent competition of medications. The phosphine reducing agent P 3001 is capable of dissolving disulfide bonds of mucin proteins, thereby decreasing the viscoelasticity of mucus. Theoretically, this allows for improved mucus clearance, lessening the incidence of mucus plugging associated airflow obstruction Indeed, AOE challenged WT mice that received aerosolized P 3001 showed decreased Rrs changes in response to IV MCh than AOE challenged non mucolytic treated WT mice The mucolytic treated, AOE challenged WT mice showed a lower respiratory system resistance compared to the AOE challenged untreated mice Linear regression on log (x) transformed data showed that the slopes of the AOE challenged untreated WT mice were statistically significant (p value < 0.05) by one

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51 comparison tests to all other groups. This confirms that P 3001 was protective against AHR in these mice The Rrs levels of the treate d mice were similar to the Muc5ac / mice, which have previously shown to be protected against AHR Importantly, given the striking overlap between these two findings, my data suggest that the mucolytic effect induced pharmacologically (acute reversal) is as efficacious as complete absence of Muc5ac driven by gene deficiency (genetic prevention) Given that my data show that the mucolytic P 3001 is an effective reducing agent in a mouse model of allergic asthma in mice, it is enticing to speculate that fu ture treatments could employ the use of a mucolytic compound in the treatment of asthma in humans My results show that not o nly were the mice protected against AHR, but also demonstrated the breakdown of mucin proteins with western blots, and decrease d mucus plugging illustrated by histology. Mucolytics may p rove to be a useful compound to recruit in the treatment of human asthma.

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52 CHAPTER III FUTURE DIRECTIONS Future studies with mucus altering compounds will necessitate an accurate meth od of mucus quantification. One possibility is to use m icroscope imaging of histology for mucus quantification. Another approach would be the use of carbon nanoparticles delivered by aerosol. The use of fluorescing as well as black carbon nanoparticles wer e attempted in this study The techniques of both methods will need to be refined further to elicit reliable, unbiased data. Microscopy Methods The following procedure was employed for this purpose. Slides were initially loaded into microscope and the over view area was defined to include all stained lung sections. I n order to attain a random sample, each section from the overview was initially visualized at 4 times magnification specimen objective. Field of vision was arranged to be the approximate center o f the tissue with image unfocused so that no structures could be identified Magnification was then increased to 10 times and the image was taken. There were approximately 45 images per slide. Two duplicates of each image were created to facilitate count ing of both, airway versus parenchyma, and the presence or absence of mucus in the bronchi and bronchioles. Using a digital reticle, a grid of precise densit y overlaid on images areas of airways and extracellular mucus were quantified using count and measure tools in the c ell S ens software (Olympus, Center Valley, PA). For the airway versus parenchyma counts, a 250 grid was overlaid on image ( Fig. 21 ). Intersections of the grid were declared

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53 Figure 21 Airway versus Parench yma grid for mucus quantification. Initial counts of airway versus parenchyma were done using the digital reticule and count and measure features on the Olympus BX63 microscope. A 250/250 grid superimposed on image showing counts for parenchyma ( blue cross es ), and airway ( green crosses ).

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54 c ell S ens software maintains a tally of the percentages of each class. above grid, were given a closer inspection and counts were made in the region containing the airway. Intersections that contained mucus in bronchi were labeled with the red cross and airway lumen in this region was labeled with a purple cross. Intersections that contained mucus in a bronchiole, were labeled with a salmon cross and airway lumen in this region was labeled with a yellow cross ( Fig. 22 ). Terminal bronchioles and smaller airway/alveolar spaces were not tallied as previous investigations have revealed that they are not lined by mucin producing cells and thus rarely contain any secreted mucus. Microscopy Results Marks are then quantified to relative percentages. The quantification results of an AOE challenged, untreated BALB WT mouse (n = 1), and that of the AOE challenged, P 3001 treated BALB WT mouse (n = 1) are shown ( Table 2 ). Results show that the AOE challenged, non mucolytic treated WT mice had 46% occlusion in the bronchi, versus only 16% occlusion in the P 3001 treated WT mice The combined counts for airway occlusion for the bronchi plus bronchioles showed that 25% of the airways were occluded with the challenged untreated mouse, compared to the P 3001 treated who only had 10% occlusion. Also of note, the P 3001 treated mouse had a greater percentage of mucus in the bronchi (16%) versus in the bronchioles (3%). This may indicate that mucociliary clearance was effectively mobilizing mucus towar d the larger airways for clearance.

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55 Figure 22 Airway versus mucus quantification grid. Image showing a 50/50 grid used for quantification of airway mucus in images previously identified to contain an airway Interse ctions with mucus in a bronchi we r e labeled with a red cross, bronchi wit h clear airway lumen we re labeled with a purple cross Intersections with a bronchiole containing mucus we re labeled with a salmon cross and bronchiole clear airway lumen with a yellow cross.

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56 Table 2 Q uantitative assessments of mucus plugging for an AOE challenged, untreated mouse and an AOE challenged, P 3001 treated mouse. Total Lung AOE Challenged U ntreated 912 Total Counts AOE Challenge d + P 3001 432 Total Counts Airway 61/912 ( 6.7% ) 44/432 ( 10.2% ) Parenchyma 881/912 ( 93.3% ) 388/432 ( 89. ) 8% Lumen vs Mucus AOE Challenged Untreated 781 Total Counts AOE Challenge d + P 3001 779 Total Counts Bronchi Positive 82/177 ( 46% ) 71/453 ( 16% ) Bronchi Negative 95/177 ( 54% ) 382/453 ( 84% ) Bronchiole Positive 116/604 ( 19% ) 10/326 ( 3% ) Bronchiole Negative 488/604 ( 81% ) 316/326 ( 97% ) Combined Bronchi & Bronchiole Positive 198/781 ( 25% ) 81/779 ( 10% )

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57 Another interesting finding is that the percentage of airways is lower for the untreated mouse in the airway versus parenchyma count. Since the total number of larger airways does not change, this may indicate an increase in parenchymal airspaces (hyperinflation from a irflow obstruction) making the airway percentage smaller relative to the parenchymal airspaces. This is a developing system and the technique is being refined. The end goal of this procedure is to have an unbiased, random, and efficient method of mucu s quantification. Carbon Nanoparticle Methods Fluorescent carbon nanopa rticles are emerging as a useful labeling technique, and have shown superior brightness and photostability compared to conventional molecular probes (Bhunia et al. 2013). Initial research into the use of nanoparticles to assess mucociliary clearance have s hown that mucociliary clearance of particles is independent of size, shape and charge of the particles, probably due to the lack of penetration of particles through the mucus blanket (Kirch et al. 2011). Nonetheless, the use of aerosolized nanoparticles is of current interest for both diagnostic and therapeutic research (Yang et al 2008). The use of fluorescent and black carbon nanoparticles was explored with this study, but results were mixed. Carbon nanoparticles were employed with intubated mice. Particl es were delivered, and mucociliary clearance was assessed at different time points (time 0 60 minutes) using bronchoalveolar lung fluid (BALF). After samples were obtained, they were scanned with a Bio Tek plate reader (Bio Tek Instruments, Winooski, VT) I was unable to ascertain any significant differences with this technique, as all samples failed to yield signals above the autoflourescence of lung lavage fluid. It is unclear if the lack of signal was due to

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58 aerosol delivery, low yields from lavage, or w eak fluorescence of the particles (<10% quantum yield). In addition to the above techniques, black carbon nanoparticles were also investigated using the same surgical procedures previously described (see methods Chapter II ). In a pilot study, observed ro bust deposition of particles ( Fig. 23 ). However, since the particles were non fluorescent, I sought to determine their localization and transport by microscopy. Mice were ventilated and treated with saline or P 3001 and MCh exactly as described for the AHR studies above (again, see Methods). Delivery of black CNP particles was accomplished with the use of the flexiVent. 150 nm black CNP were diluted to 25 mg/ml to facilitate aerosol delivery. Animals received six 10 second doses for a total delivery of ~ 4.945 mg of particles. Mice were then euthanized and lungs fixated in methacarn and prepared for histology as before. Initial results have shown that black carbon nanoparticles are visible on the airway surfaces before being embedded in paraffin. Unfortu nately, after the paraffin embedding and staining processes, nanoparticles are largely absent on the slides. This may be due to the rehydration/dehydration process with xylene and graded alcohols that the slides undergo during the staining process, as well as the poor adhesion of the neutrally charged particles on the positively charged glass microscope slides. Further experiments will help to refine a technique that will mark the progress of mucociliary clearance. Perhaps other methods such as the use of U lex Europaeus Agglutinnin I (UEA I), a lectin that binds to the glycoproteins on the mucins could be used to mark mucociliary clearance (Roney et al 2011). Finding a useful technique that is random, unbiased, and reliable in the laboratory setting would be

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59 Fig. 23 Left lung filet illustrating deposition of black carbon nano particles (CNP) at time 0

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60 helpful to assess the efficacy of pharmacological compounds on improving mucociliary transport in the future. Conclusions and Impact of Findings In summary, using a mouse model of human asthma, I have demonstrated that a novel mucolytic compound P 3001 was capable of reversing AHR and mucus plugging in mice via disruption of mucin polymers. I have also shown that reversing mucus hypersecretion is a potential therapeutic strategy for allergic asthma As new research regarding mucin proteins is emerging, concurrent research in pharmacology should be undertaken. Since mucus is prominent in asthma episodes and a key feature of fatal a sthma attacks, research needs to be continued using new drugs, specifically mucolytics. As noted by Sidebotham and Roche in 2003, asthma deaths are a persistent and preventable mortality. The use of a phosphine based reducing agent could be an effective t herapeutic strategy for reducing the viscoelasticity of mucus in the airway, thereby, reducing plugging. Hence, phosphine based reducing agents could be effective in reducing fatalities as well improving quality of life for patients with mild to moderate d isease. Mucolytics may also prove useful for other diseases with impaired mucociliary clearance such as, COPD, CF, and ciliary dyskinesia.

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65 Krefft, S. D., Meehan, R., & Rose, C. S. (2015). Eme rging spectrum of deployment related respiratory diseases. Current Opinion in Pulmonary Medicine, 21 (2), 185 192. doi:10.1097/mcp.0000000000000143 Kuyper, L. M., Par, P. D., Hogg, J. C., Lambert, R. K., Ionescu, D., Woods, R., & Bai, T. R. (2003). Charac terization of airway plugging in fatal asthma. The American Journal of Medicine, 115 (1), 6 11. doi:10.1016/s0002 9343(03)00241 9 Lu, W. (2004). Effects of dexamethasone on Muc5ac mucin production by primary airway goblet cells. AJP: Lung Cellular and Mole cular Physiology, 288 (1). doi:10.1152/ajplung.00104.2004 Lundgren, J. D., Kaliner, M., Logun, C., & Shelhamer, J. H. (1988). Dexamethasone Reduces Rat Tracheal Goblet Cell Hyperplasia Produced by Human Neutrophil Products. Experimental Lung Research, 14 (6 ), 853 863. doi:10.3109/01902148809087849 Mcgovern, T. K., Robichaud, A., Fereydoonzad, L., Schuessler, T. F., & Martin, J. G. (2013). Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique. Journal of Visualized Experiments JoVE, (75). doi:10.3791/50172 Mushaben, E. M., Brandt, E. B., Hershey, G. K., & Cras, T. D. (2013). Differential Effects of Rapamycin and Dexamethasone in Mouse Models of Established Allergic Asthma. PLoS ONE, 8 (1). doi:1 0.1371/journal.pone.0054426 Ordoez, C. L., Khashayar, R., Wong, H. H., Ferrando, R., Wu, R., Hyde, D. M., Fahy, J. V. (2001). Mild and Moderate Asthma Is Associated with Airway Goblet Cell Hyperplasia and Abnormalities in Mucin Gene Expression. Am J Respir Crit Care Med American Journal of Respiratory and Critical Care Medicine, 163 (2), 517 523. doi:10.1164/ajrccm.163.2.2004039 Petk, F., Hantos, Z., Adamicza, Asztalos, T., & Sly, P. D. (1997). Methacholine induced bronchoconstriction in rats: E ffects of intravenous vs. aerosol delivery. The American Physiological Society Rogers, D. (2004). Airway mucus hypersecretion in asthma: An undervalued pathology? Current Opinion in Pharmacology, 4 (3), 241 250. doi:10.1016/j.coph.2004.01.011 Rogers, D. F. (2003). The airway goblet cell. The International Journal of Biochemistry & Cell Biology, 35 (1), 1 6. doi:10.1016/s1357 2725(02)00083 3 Roney, C. A., Xu, B., Xie, J., Yuan, S., Wierwille, J., Chen, C., Chen, Y. (2011). Rh I UEA 1 polymerized lipo somes target and image adenomatous polyps in the APC(Min/+) mouse using optical colonography. Retrieved from http://europepmc.org/articles/PMC3415799

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66 Roy, M. G., Livraghi Butrico, A., Fletcher, A. A ., Mcelwee, M. M., Evans, S. E., Boerner, R. M., Evans, C. M. (2013). Muc5b is required for airway defence. Nature, 505 (7483), 412 416. doi:10.1038/nature12807 Scireq. (2016). Benefits of using the flexivent. Retrieved from http://www.scireq.com/flexivent Shalaby, K. H., Gold, L. G., Schuessler, T. F., Martin, J. G., & Robichaud, A. (2010). Combined forced oscillation and forced expiration measurements in mice for the assessment of airway hyperresponsi veness. Respiratory Research, 11 (1). doi:10.1186/1465 9921 11 82 Sheehan, J. K., Richardson, P. S., Fung, D. C., Howard, M., & Thornton, D. J. (1995). Analysis of respiratory mucus glycoproteins in asthma: A detailed study from a patient who died in statu s asthmaticus. American Journal of Respiratory Cell and Molecular Biology Am J Respir Cell Mol Biol, 13 (6), 748 756. doi:10.1165/ajrcmb.13.6.7576713 Sidebotham, H. J., & Roche, W. R. (2003). Asthma deaths; persistent and preventable mortality. Histopathol ogy, 43 (2), 105 117. doi:10.1046/j.1365 2559.2003.01664.x Soma, L. R. (1983). Anesthetic And Analgesic Considerations In The Experimental Animal. Annals of the New York Academy of Sciences, 406 (1 The Role of A), 32 47. doi:10.1111/j.1749 6632.1983.tb53483.x Takami, S., Mizuno, T., Oyanagi, T., Tadaki, H., Suzuki, T., Muramatsu, K., Arakawa, H. (2012). Glucocorticoids Inhibit MUC5AC Production Induced by Transforming Growth Factor in Human Re spiratory Cells. Allergology International, 61 (3), 451 459. doi:10.2332/allergolint.11 oa 0411 Thornton, D. J., & Sheehan, J. K. (2004). From Mucins to Mucus: Toward a More Coherent Understanding of This Essential Barrier. Proceedings of the American Thoracic Society, 1 (1), 54 61. doi:10.1513/pats.2306016 Thornton, D. J., Rousseau, K., & Mcguckin, M. A. (2008). Structure and Function of the Polymeric Mucins in Airways Mucus. Annual Review of Physiology Annu. Rev. Physiol., 70 (1), 459 486. doi:10.1146/ annurev.physiol.70.113006.100702 Yamabayashi, C., Koya, T., Kagamu, H., Kawakami, H., Kimura, Y., Furukawa, T., Narita, I. (2012). A Novel Prostacyclin Agonist Protects against Airway Hyperresponsiveness and Remodeling in Mice. American Journal of R espiratory Cell and Molecular Biology, 47 (2), 170 177. doi:10.1165/rcmb.2011 0350oc Yang, W., Peters, J. I., & Williams, R. O. (2008). Inhaled nanoparticles International Journal of Pharmaceutics, 356 (1 2), 239 247. doi:10.1016/j.ijpha rm.2008.02.011

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67 Yuan, S., Hollinger, M., Lachowicz Scroggins, M. E., Kerr, S. C., Dunican, E. M., Daniel, B. M., Fahy, J. V. (2015). Oxidation increases mucin polymer cross links to stiffen airway mucus gels. Science Translational Medicine, 7 (276). d oi:10.1126/scitranslmed.3010525 Zdanowicz, M. M. (2007). Pharmacotherapy of Asthma. American Journal of Pharmaceutical Education, 71 (5), 98. doi:10.5688/aj710598

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68 APPENDIX A Protocols for FlexiVent and Western Blots Protocol for flexiVent FlexiVent In Vivo Asthma Study Protocol With IV Methacholine I: 1 day to 1 week prior to experiment Click on flexiware icon to launch welcome dialog We Study Definition and P lanning C reate new study click create a new study button. Fill in the required info and click next until you reach step 6: confirmations. 1. Title and status: A ssign the st udy a title, enter a name of the p erson responsible for the study and select a status for the study (Identified or Scheduled ) Next 2. Objective and Hypothesis : Next 3. Protocol: Next 4. Subject groups: New button Subject group dialogue nter the title of your group status (started or not started) subject quantity in the group and details 5. Assign subject: Create new subject icon Subject Detail D ialogue Fill in t he unique subject identifier species and the subject weight must be completed; the remaining fields (e.g. strain birthdate ) are optional assi gn the subject to group membership (found below subject Next Finish. 6. Confirmation Supplies needed for experiment: 1 x18 gauge beveled blunt tip cannula /mouse 1 piece of silk or polyester thread tie (15 20 cm long) 1 ml syringes (urethane, succinyl choline for i.p. bolus methacarn, saline flushes for IV insertion, and to collect BAL) 3 ml syringe for IV infusion of succinylcholine and methacholine Tubes for BALF collection ( 6 x 1 .5 ml, 1 x 1 .0 ml per mouse, 2 cryovials per mouse ) Scintillation vials filled with methacarn for fixation (1 per mouse) 20 gauge shielded IV catheter to cannulate the inferior vena cava Saline

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69 succinylcholine chloride (0.2 ml of a 2 0 m g/ml solution ) for i.p. injecion and (50 ) for IV infusion methacholine ( solution ) added to succinylcholine for IV infusion urethane ( 2g/kg of a 0.01 ml/g solution ) for i.p. injection surgical instruments p 200 and p1000 pipettes and tips II: On the day of experiment Prepare the styroform setup: mouse) two paper towels and tape for securing mice and platform Fill and prime 3 ml syringe with succinylcholine chloride and methacholine mixture Prepare supplies for IV insertion Set up syringe mini pump (rate 4 l/g/min) Prepare syringes for i.p. urethane and succinylcholine administration, saline for IV flushes, and methacarn for fixation Fill scintillation vials with methacarn and label then place on ice Cut lengths of silk thread (one for each mouse) Flexiware Experimentation session At this point the program will prompt you with series of dialogues and wizard Study selection : Select your study that you predefined and saved Template properties : Mouse (AN/EKG) custom Flexiware module : 1.55ml OK verify the module dialogue (flexivent FX1) OK Add module with aeroneb mount Attach aeroneb Subject site assignment : drag and drop the animal that you going to use from right box to left OK Subject weight confirmation : weigh the mouse and enter the current weight Auto calibration and warm up Prime the nebulizer : connect the nebulizer Place 100 l Saline hold it u p and click next you should see aerosol emerging from the bottom of aeroneb, and when finish clean excess PBS and reattach to base Calibration: Make sure that each tube and nebulizer is dry, and there are no leaks. Channel calibration:

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70 Step 1: (Channel Selection) both Cylinder pressure (step 2 5 ) and Airway pressure (step 6 9 ) NEXT Cylinder pressure calibration Step 2: First va lue: Zero A tmosphere (0 mmH 2 O): Leave the Y tubing open to atmosphere NEXT Step 3: Second Value: Known Pressure : Connect the SCIREQ manometer to the Y tubing apply pressure over 300 mmH 2 O mark via syringe (The indicator panel on the right pressure bar will turn from red to green) mmH 2 O (**this v alue can be enter if you click next at other pressure value other than 300 mmH 2 O. Step 4: Target value input: Enter 300 mmH 2 O NEXT Step 5 : Channel calibration results : ra nges should be Max value = 80 8 c mH 2 O and Min value = 80 8 c mH 2 O range 160 16 Airway pressure calibration Step 6 : First value: Zero atmosphere (0 mmH 2 O): Leave the Y tubing open to atmosphere NEXT Step 7 : Second Value: Known Pressure : Connect the SCIREQ manometer to the Y tubing apply pressure over 300 mmH 2 O mark via syringe (This will indicator panel on the right pressure bar will turn from red to green mmH 2 O (**this value can be enter if you click next at other pressure value other than 300mmH 2 O. Step 8 : Target v alue input: (Enter known value for the first point) NEXT Step 9 : Channel calibration results: ra nges should be Max value = 80 8 c mH 2 O and Min value = 80 8 c mH 2 O range 160 16 Click Finish to exit channel calibration Tube Calibration: Should be performed before EACH new subject in an experimentation session NEXT Step 2: Perturbation selection: TLC Snapshot 150 and Quick Prime 3 are to be selected NEXT Step 3: Closed Preparation: Place tracheal cannula on ventilator Block the tracheal cannula with your thumb. NEXT Step 4: Closed Calibration: Keep cannula blocked while the system performs closed calibration Step 5: Open Preparation: Remove thumb from the tracheal cannula so it is open to the air NEXT Step 6: Open Calibration: Keep cannula open while the system performs open calibration

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71 Check values: Prime TLC R S (shunt resistance) 2000 Snapshot (tube resistance) should be lower that baseline resistance 0.38 c mH 2 O Once the calibration s are done, you will be prompted t o begin the default ventilation. Do not start ventilation yet. First, perform surgery on mouse. Animal Preparation and Performing the Experiment Anesthetize first mouse 10 of urethane (concentration: 2 00mg/ml dosage; 2.0g /kg ) To tracheo stomized : Remove strap muscles to expose trachea Separate esophagus away from trachea using forceps Slip thread under the trachea as a half loop Cut the thread in the middle leaving two equal size pieces Make a small cut (~0.1in) on the trachea with micro scissors Slip in cannula (half its length inside the trachea) and tie the thread in order to secure cannula. Start ventilation C onnect tracheal cannula to ventilator, while keeping cannula and mouse in the neutral position above the heating pad then secure mouse to the platform Inject mouse with 0.2 ml succinylcholine chloride (10 g/ml) i.p. Open the abdomen and identify the inferior vena cava (IVC) Cannulae the IVC with the 20 gauge sheilded IV catheter and confirm success by observing blood return in the catheter After flushing the catheter with saline, connect IV tubing insuring that there are no air bubbles in the line Start the syringe pump at rate of 4 l/ k g/min. Check for adequate paralysis. Look at the monitor panels on the bottom of flexivent screen look for even peaks and valleys Load Script: Automation load scripts select script (e.g. Leslie Inhaled dose response with IV infusion ) double click script name in window or select alternate start with automation start script Begin with the baseline ( 4 l/kg/min of methacholine ), when prompted

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72 Doses of IV succinylcholine and methacholine mixture : 4 l/kg/min 8 l/kg/min 16 l/kg/min 32 l/kg/min 64 l/kg/min 128 l/kg/min ***ALLOW FULL RECOVERY BETWEEN DOSES*** Once you finish all doses, click cancel on the Stop O Stop the syr inge pump R emove mouse from flexivent and proceed with sample collection. If subject is to be prepared for histology, remove cannula and immediately tie off trachea Inject 0.4ml of methacarn into the intrapleural space and gently distribute by rotating After 20 minutes, excise heart and lungs from the chest cavity and place in the scintillation vial filled with methacarn Place specimens in the 40 C refrigerator Between mice, the following should be performed Detach nebulizer and Y tubing from their bas e and dry condensation with Kimwipes. Blow compressed through the Y tubing, to flush out any remaining condensation. Reload succinylcholine and methacholine for next mouse in the syringe pump assign subj ect and load the next mouse to subject assignment Start channel calibration o Closed Preparation: Place tracheal cannula on ventilator. Block the tracheal cannula with your thumb. next o Closed Calibration: Keep cannula blocked while the system performs closed calibration o Open Preparation: Remove thumb from the tracheal cannula so it is open to the air next o Open Calibration: Keep cannula open while the system performs open calibration. o Verify the values R r s value Clean Up Remove the Y tubing adapt or. Connect a quick connect to the expiratory port on the front of the module (right). Place a second quick connect in the air/gas exhaust port on the back of the module (bottom) Place a beaker at the exit of the tubing connected to air/gas exhaust port. Using 10 ml syringe, run 6 x 10 ml isopropyl alcohol into the tubing that is attached to the expiratory port.

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73 Repeat previous step as needed. Flush out any liquid remaining in the lines by blowing air through the tubing attached to the front expiratory po rt. Clean Y tubing with water and dry with kimwipes Remove quick connects. Replace Y tubing adapter Attach the test load (1 ml syringe) to Y tubing Start default ventilation and let it run for approximately 15 min to ensure that the expiratory line is com pletely dry. After 15min, stop default ventilation Close program Reviewing data/Export Data export Data export is divided into two portions in flexiWare: Export Scenarios and Data Export. Export Scenarios are containers in which an export strategy is described. They are generic and define only the type of data to be exported (e.g. subject data, parameters) and the export type (e.g. ASCII, Excel). They are saved in the database and may be used repeatedly. Data Export delimits the actual data to be e xported (e.g. subject selection, timeframe). EXPORT SCENARIO To define a new Export Scenario select new button You must provide a unique name, the type of data you wish to export in this scenario and for flexiVent applications on ly, the relevant perturbations. There are four types of data that can be exported: Subjects: exports all of the subject details including statistics regarding data collection Completed with the subject. Dataset Parameters: exports analyzed parametric outcomes from datasets.

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74 Dataset Signals: exports analyzed non parametric outcomes from datasets; these are often intermediary results which are used to generate the final dataset parameters. Strea m Data: Exports continuous streams of data When selecting dataset parameters or dataset signals, you must enter the name of the data to be exported. The names entered must match the names that are defined for each of the outcomes on the applicable analy zer page(s) of the Perturbation Properties dialogue. Specify whether you wish to export the data to Microsoft Excel or ASCII format. This screen also gives you the opportunity to include excluded datasets, event comments and user markers Click Finish DATA EXPORT Once you have created your export scenario(s), you can proceed to data export by selecting File Export Export Data. This displays the Data Export Wizard that allows you to export your data in five easy steps. 1. Select the Export Scenario(s) yo u would like to use You can export data using multiple data export scenarios simultaneously. 2. Select the subject(s) for which you wish to export data. The subject filter at the top of the screen allows you to select All subjects, those marke d as Valid, those marked as Invalid or Selected subjects. If you choose Selected subjects you can multi select by holding down the Ctrl key as you click on each subject. 3. Specify the export timeframe for data export. The following three choices ar e available: Entire study timeframe exports all data belonging to the current study. Between specific events allows you to select a start and end event. Based on subject time allows you to export between specified times using the relative t ime scale i.e. all subjects start at time 0, for example, this option would allow you to export the first 10 minutes of data for each subject. 4. Specify the export destination folder. If you leave it set to the default, your data is exported to C: \ Users \ Public \ Documents \ SCIREQ \ flexiWare \ [St udy Name]. The file(s) from the export will be named according to the selected export scenar io. On this screen you can also specify how the software should proceed if filename for t he exported data already exist 5. Click Export Protocol for Western Blots Western Blot Protocol for Mouse Muc5b from BALF Licor Materials needed:

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75 LE Agarose Gene Mate Cat No. E 3120 500 Gel unit (Recycling B3 unit or the Large A series unit from the Lozupone Lab) Gel/Running Buffer (2L) 1X TAE + 0.1% w/v SDS (add 1600ml H2O +40ml 50X TAE bring to 2L mix take out 20ml of solution +20ml 10% SDS mix well) Glassware: cleaned well using S parkleen rinsed in DI Water PVDF Membrane : IPFL00010 Immobilon FL PVDF, 0.45 m (membrane cut to 125 x 140 mm) 100% MeOH Paddle tweezers (2 pairs) Pencil to mark membrane Sereological pipette 5 mL to roll out gel Vacuum flasks: with in H 2 O vacuum g uage(s) and regulator(s) Blot Paper : BIO RAD thick blot filter paper (un cut) Cat no. 1703955 DTT: Amresco CAS Number: 3483 12 3 (dry powder in 20C) Odyssey Blocking Buffer (TBS) 125 mL [P/N 927 50100] Working Solutions: 50% Odyssey blocking solution and 50% 1X TBS for blocking or 50% 1X TBST for the antibody dilutions/incubations (~40 ml will cover blots) 1 o Ab : rabbit anti mouse Muc5b ( 20C aliquoted undiluted Ab; use at 1:5,000) 2 Ab : IRDye 680RD Goat anti Rabbit (Lot# C41029 03; Stored undiluted at 4C; used at 1:15,000 dilution) 1 X TBS : 12.114 g Tris HCl + 87.66 g NaCl, pH 8.0 and make up to 1 L with ddH2O 1 X TBST : 1x TBS + 0.1% Tween 20 10% SDS stock solution 20X SSC : 175.32g NaCl + 88.2 g Na Citrate, fill to 1L w ith dd H 2 O, pH to 7.0 Working solution (per liter) : 4x SSC: 200 ml of 20x SSC + 800 ml ddH 2 O

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76 1X TBS: ( 12.114 g Tris HCl + 87.66 g NaCl, Adjust pH to 8.0 and make up to 1 L with ddH2O) 1 X TBST : 1x TBS + 0.1% Tween 20

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77 Gel Preparation Template : Optimization Tips (from LI C OR ) 1. To avoid background speckles on blots, use high quality ultrapure water for buffers. 2. Rinsing previously used incubation boxes with methanol can reduce background contamination of future blots 3. Never perform incubations or washes in dishes that have been previously used for Coomassie staining. 4. Membranes should be handled only by their edges, with clean forceps 5. Always pour off antibody solution and washes from the same corner of the box to ensure complete removal of previous solutions. 6. After handling membranes that have been incubating in antibody solutio ns, clean forceps thoroughly with DI water and/or methanol, then rinse with distilled water. X 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 X Sample ID BAL vol Offest vol. Plate well (optional)

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78 7. Do not wrap the membrane in plastic when scanning.

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79 Procedures : WESTERN BLOT DAY 1 Prepare running buffer and cool in the cold room. Make 0.9% agarose 0.1% SD S TAE 14x13 cm gel: Weigh (record weight) and tare 500 mL flask (use high wt capacity balance) Add 100 mL ddH2O + 1.62g agarose to flask Cover flask opening with KIM wipe to avoid spill over Boil: set timer on the microwave to 3 min let run for 1 min stop and gently shake stop every 20 30 sec thereafter Cool for a couple of min Heat additional 100 ml of ddH2O for ~1 min in microwave Add 3.6 mL 50x TAE and 1.8 mL 10% SDS (along the glass to prevent bubbling) Reweigh (record weight to make sure appro priate volume lost is added back; 1 mL H2O = 1 g) Fill with hot ddH2O until 180 g Use 1.5 mm 20 well comb side to cast the gel Thaw BAL samples in ice/water slurry. (Add protease inhibitor, 10% of sample volume.) Label 1.5ml tubes with arbitrary numbers ( Swirl BAL samples with pipette tip, and add BALF and H2O to labeled 1.5 ml tubes according to the normalization analysis for the total of 25 ul. Add 5 ul of loading buffer (nominally 10X buffer) Spin down the tubes for briefly (3 5 sec) on b enchtop microfuge Fill gel unit with running buffer. Load gel (DO NOT use 2 outside lanes: these will not transfer ). Run overnight at 42 V in cold room or deli cooler. WESTERN BLOT DAY 2 Photograph gel with a ruler to see marker location Soak gel in 4x SSC + 10mM DTT (0.15425g DTT per 100mL of 4x SSC) at RT for 20 min with gentle shaking (10 20 RPM) Activate PVDF FL in 100% MeOH for 15 sec Dump MeOH and add 50 100 ml 4x SCC Gently shake (10 20 RPM) >5 min. Assemble transfer apparatus: sponge filt er paper Saturate filter paper with 4X SSC prior to placing PVDF membrane on top

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80 Finish assembling the transfer apparatus PVDF membrane (roll out any visible bubbles with serological pipette and keep membrane wet with 4x SSC), transfer mask, gel. Once the gel is in place make sure there are no bubbles underneath by rolling them out with a stereological pipette Turn on vacuum and set to 10 15 inches H 2 O Pour 4X SSC to submerge the gel. Make sure there are NO leaks! Do not leave the tra nsfer unit running unattended. Remove PVDF membrane with clean tweezers and rinse for 2 Do not let PVDF to dry Place membrane in incubation box and block the membrane in Odyssey Blocking buffer working solution for 1 hr on rocker (set rocker to 1 2) Prepare 1 o antibody dilution o 20 ml Odyssey blocking buffer o 20 ml TBST o 8 uL 1 o Ab rabbit anti mouse Muc5B o Mix well o Add to the membrane WESTERN BLOT DAY 3 Bring membrane to RT. Wash membranes o Pour off primary antibody solution o Rinse membrane with 1X TBS T (0.1% Tween 20), dump immediately o Cover blot with 1X TBS T (0.1% Tween 20) o (set rocker to 1 2) o Pour off wash solution o Repeat for a total of 3 4 washes Prepare 2 o antibody dilution o 20 ml Odyssey blocking buffer o 20 ml TBST o 40 ul 10% SDS stock solution (final conc. = 0.01% SDS) o 2.7 ul Secondary Antibody (1:15,000) o Mix well o Add to the membrane

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81 Protect the membrane from light during incubation u sing box or foil. Incubate @ RT for 1hr with gentle shaking Protect membrane from light during washes! Wash membranes o Pour off primary antibody solution o Rinse membrane with 1X TBS T (0.1% Tween 20), dump immediately o Cover blot with 1X TBS T (0.1% Tween 2 0) o (set rocker to 1 2) o Pour off wash solution o Repeat for a total of 3 4 washes Rinse membrane with 1X TBS to remove residual Tween 20. Membrane is now ready to be imaged

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82 APPENDIX B FlexiVent Raw Data Group Dose Rrs Unchallenged, n = 7 4 1.697806 Unchallenged, n = 7 4 1.485296 Unchallenged, n = 7 4 1.598057 Unchallenged, n = 7 4 0.738117 Unchallenged, n = 7 4 1.21963 Unchallenged, n = 7 4 0.841489 Unchallenged, n = 7 4 0.843196 Unchallenged, n = 7 8 1.613589 Unchallenged, n = 7 8 1.770998 Unchallenged, n = 7 8 2.127709 Unchallenged, n = 7 8 0.818583 Unchallenged, n = 7 8 1.018494 Unchallenged, n = 7 8 1.032738 Unchallenged, n = 7 8 1.956596 Unchallenged, n = 7 16 1.626127 Unchallenged, n = 7 16 1.767881 Unchallenged, n = 7 16 2.008483 Unchallenged, n = 7 16 0.869276 Unchallenged, n = 7 16 1.14151 Unchallenged, n = 7 16 Unchallenged, n = 7 16 2.467539 Unchallenged, n = 7 32 2.585368 Unchallenged, n = 7 32 1.929785 Unchallenged, n = 7 32 2.973254 Unchallenged, n = 7 32 0.930825 Unchallenged, n = 7 32 5.405394 Unchallenged, n = 7 32 2.190499 Unchallenged, n = 7 32 2.974648 Unchallenged, n = 7 64 4.002827 Unchallenged, n = 7 64 2.280709 Unchallenged, n = 7 64 3.689791 Unchallenged, n = 7 64 0.977061 Unchallenged, n = 7 64 Unchallenged, n = 7 64 3.036626 Unchallenged, n = 7 64 3.049514 Unchallenged, n = 7 128 5.931221 Unchallenged, n = 7 128 2.578164 Unchallenged, n = 7 128 3.689791

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83 Unchallenged, n = 7 128 1.067326 Unchallenged, n = 7 128 Unchallenged, n = 7 128 3.138034 Unchallenged, n = 7 128 4.626993 BALB WT AOE, n = 13 4 1.573098 BALB WT AOE, n = 13 4 1.797719 BALB WT AOE, n = 13 4 1.839151 BALB WT AOE, n = 13 4 1.216133 BALB WT AOE, n = 13 4 1.546626 BALB WT AOE, n = 13 4 1.227731 BALB WT AOE, n = 13 4 1.484908 BALB WT AOE, n = 13 4 1.471928 BALB WT AOE, n = 13 4 1.454716 BALB WT AOE, n = 13 4 1.80948 BALB WT AOE, n = 13 4 1.509645 BALB WT AOE, n = 13 4 1.225105 BALB WT AOE, n = 13 4 1.880144 BALB WT AOE, n = 13 8 0.912943 BALB WT AOE, n = 13 8 3.739564 BALB WT AOE, n = 13 8 2.645449 BALB WT AOE, n = 13 8 1.472816 BALB WT AOE, n = 13 8 1.327337 BALB WT AOE, n = 13 8 0.773229 BALB WT AOE, n = 13 8 1.779645 BALB WT AOE, n = 13 8 4.652586 BALB WT AOE, n = 13 8 4.785667 BALB WT AOE, n = 13 8 2.144764 BALB WT AOE, n = 13 8 3.641887 BALB WT AOE, n = 13 8 1.153776 BALB WT AOE, n = 13 8 2.264587 BALB WT AOE, n = 13 16 1.355516 BALB WT AOE, n = 13 16 3.70446 BALB WT AOE, n = 13 16 1.839151 BALB WT AOE, n = 13 16 9.660468 BALB WT AOE, n = 13 16 1.744475 BALB WT AOE, n = 13 16 0.901226 BALB WT AOE, n = 13 16 4.222582 BALB WT AOE, n = 13 16 3.119045 BALB WT AOE, n = 13 16 9.653567 BALB WT AOE, n = 13 16 8.163266 BALB WT AOE, n = 13 16 11.67767 BALB WT AOE, n = 13 16 2.09324 BALB WT AOE, n = 13 16 6.802396

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84 BALB WT AOE, n = 13 32 1.615039 BALB WT AOE, n = 13 32 12.56136 BALB WT AOE, n = 13 32 1.751621 BALB WT AOE, n = 13 32 10.4606 BALB WT AOE, n = 13 32 3.405692 BALB WT AOE, n = 13 32 3.046979 BALB WT AOE, n = 13 32 5.138819 BALB WT AOE, n = 13 32 6.355516 BALB WT AOE, n = 13 32 10.20448 BALB WT AOE, n = 13 32 6.64443 BALB WT AOE, n = 13 32 18.37788 BALB WT AOE, n = 13 32 3.286127 BALB WT AOE, n = 13 32 7.119519 BALB WT AOE, n = 13 64 2.26746 BALB WT AOE, n = 13 64 41.30844 BALB WT AOE, n = 13 64 3.642246 BALB WT AOE, n = 13 64 25.19357 BALB WT AOE, n = 13 64 3.559284 BALB WT AOE, n = 13 64 3.988721 BALB WT AOE, n = 13 64 BALB WT AOE, n = 13 64 BALB WT AOE, n = 13 64 16.89222 BALB WT AOE, n = 13 64 16.6267 BALB WT AOE, n = 13 64 27.31606 BALB WT AOE, n = 13 64 3.884046 BALB WT AOE, n = 13 64 10.5643 BALB WT AOE, n = 13 128 5.324903 BALB WT AOE, n = 13 128 87.22773 BALB WT AOE, n = 13 128 5.742002 BALB WT AOE, n = 13 128 42.09997 BALB WT AOE, n = 13 128 5.576558 BALB WT AOE, n = 13 128 5.516759 BALB WT AOE, n = 13 128 BALB WT AOE, n = 13 128 BALB WT AOE, n = 13 128 16.87386 BALB WT AOE, n = 13 128 25.99337 BALB WT AOE, n = 13 128 BALB WT AOE, n = 13 128 13.79561 BALB WT AOE, n = 13 128 17.65765 Muc5ac KO AOE, n = 4 4 1.99926 Muc5ac KO AOE, n = 4 4 1.402983 Muc5ac KO AOE, n = 4 4 2.209586 Muc5ac KO AOE, n = 4 4 1.410925

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85 Muc5ac KO AOE, n = 4 8 1.896724 Muc5ac KO AOE, n = 4 8 2.349068 Muc5ac KO AOE, n = 4 8 1.921075 Muc5ac KO AOE, n = 4 8 1.775477 Muc5ac KO AOE, n = 4 16 2.520983 Muc5ac KO AOE, n = 4 16 2.243595 Muc5ac KO AOE, n = 4 16 1.819613 Muc5ac KO AOE, n = 4 16 2.262319 Muc5ac KO AOE, n = 4 32 2.632477 Muc5ac KO AOE, n = 4 32 3.13292 Muc5ac KO AOE, n = 4 32 2.195089 Muc5ac KO AOE, n = 4 32 2.934794 Muc5ac KO AOE, n = 4 64 2.635094 Muc5ac KO AOE, n = 4 64 5.007348 Muc5ac KO AOE, n = 4 64 6.351069 Muc5ac KO AOE, n = 4 64 7.515857 Muc5ac KO AOE, n = 4 128 2.947929 Muc5ac KO AOE, n = 4 128 4.40801 Muc5ac KO AOE, n = 4 128 7.924337 Muc5ac KO AOE, n = 4 128 19.78577 BALB WT AOE + P 3001, n = 7 4 1.255069 BALB WT AOE + P 3001, n = 7 4 1.357685 BALB WT AOE + P 3001, n = 7 4 1.220027 BALB WT AOE + P 3001, n = 7 4 1.713947 BALB WT AOE + P 3001, n = 7 4 1.309743 BALB WT AOE + P 3001, n = 7 4 1.310526 BALB WT AOE + P 3001, n = 7 4 1.472925 BALB WT AOE + P 3001, n = 7 8 1.268291 BALB WT AOE + P 3001, n = 7 8 1.74836 BALB WT AOE + P 3001, n = 7 8 1.90285 BALB WT AOE + P 3001, n = 7 8 1.67477 BALB WT AOE + P 3001, n = 7 8 2.202705 BALB WT AOE + P 3001, n = 7 8 1.30699 BALB WT AOE + P 3001, n = 7 8 0.790453 BALB WT AOE + P 3001, n = 7 16 1.052877 BALB WT AOE + P 3001, n = 7 16 6.132445 BALB WT AOE + P 3001, n = 7 16 4.106292 BALB WT AOE + P 3001, n = 7 16 1.688387 BALB WT AOE + P 3001, n = 7 16 4.978462 BALB WT AOE + P 3001, n = 7 16 2.176566 BALB WT AOE + P 3001, n = 7 16 0.953341 BALB WT AOE + P 3001, n = 7 32 1.198506 BALB WT AOE + P 3001, n = 7 32 5.158922

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86 BALB WT AOE + P 3001, n = 7 32 5.678525 BALB WT AOE + P 3001, n = 7 32 BALB WT AOE + P 3001, n = 7 32 6.922513 BALB WT AOE + P 3001, n = 7 32 2.497327 BALB WT AOE + P 3001, n = 7 32 1.844692 BALB WT AOE + P 3001, n = 7 64 1.31687 BALB WT AOE + P 3001, n = 7 64 BALB WT AOE + P 3001, n = 7 64 6.222619 BALB WT AOE + P 3001, n = 7 64 BALB WT AOE + P 3001, n = 7 64 10.61356 BALB WT AOE + P 3001, n = 7 64 7.352855 BALB WT AOE + P 3001, n = 7 64 5.777243 BALB WT AOE + P 3001, n = 7 128 1.477769 BALB WT AOE + P 3001, n = 7 128 BALB WT AOE + P 3001, n = 7 128 11.59396 BALB WT AOE + P 3001, n = 7 128 BALB WT AOE + P 3001, n = 7 128 23.4412 BALB WT AOE + P 3001, n = 7 128 10.07361 BALB WT AOE + P 3001, n = 7 128 7.228725

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87 APPENDIX C R Code setwd("~/A.Lab Thesis.stuff/Mucolytic results") ## Unchallenged vs Challenged # read in the data first = read.csv("AHR.2.csv") # prepare the data head(first) #view firs few rows of data sum_data = ddply(first, c("Group", "Dose"), summarise, N = sum(!is.na(Rrs)), mean = mean(Rrs, na.rm=TRUE), sd = sd(Rrs, na.rm=TRUE), se = sd / sqrt(N)) summary(first) plot(Rrs) plot(first$Rrs) lm(x ~ y) # Use position_dodge to move overlapped errorbars horizontally dev.new() ggplot(sum_data, aes(x=Dose, y=mean, group=Group, color=Group)) + geom_errorbar(aes(ymin=mean se, ymax=mean+se), width=.1, position=position_dodge(0.05)) + geom_line() + geom_point() + scale_colour_manual(values = c("#DD035B","#67BB00")) + labs(title="Airway Hyperreactivity",x= expression(paste("MCh ("*mu*"g/kg/min, iv)")),

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88 y = expression(paste("Respiratory Resistance (", cmH[2]*O~"/ml/s"^" 1"*")"))) + theme(plot.title = element_text(hjust = 0.5)) + scale_y_continuous(limits=c(0, 32.5)) ### Unchallenged, challenged, and Muc5ac KO # read in the data sec = read.csv("AHR.3.csv") sec$Group = as.factor(sec$Group) sec$Dose = as.factor(sec$Dose) head(sec) #view firs few row s of data summary(sec) sum_data2 = ddply(sec, c("Group", "Dose"), summarise, N = sum(!is.na(Rrs)), mean = mean(Rrs, na.rm=TRUE), sd = sd(Rrs, na.rm=TRUE), se = sd / sqrt(N)) # U se position_dodge to move overlapped errorbars horizontally dev.new() ggplot(sum_data2, aes(x=Dose, y=mean, group=Group, color=Group)) + geom_errorbar(aes(ymin=mean se, ymax=mean+se), width=.1, position=position_dodge(0.05)) + geom_lin e() + geom_point() + scale_colour_manual(values = c("#DD035B", "chocolate 1", "#67BB00")) + labs(title="Airway Hyperreactivity",x= expression(paste("MCh ("*mu*"g/kg/min, iv)")), y = expression(paste("Airway Resistance (", cmH [2]*O~"/ml/s"^" 1"*")"))) + theme(plot.title = element_text(hjust = 0.5)) +

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89 scale_y_continuous(limits=c(0, 32.5)) #### All four # read in the data Muc_mod = read.csv("Mucolytics.F+tc.csv") Muc_mod$Group = as.factor(Muc_mod$Group) Muc_mod$Dose = as.fa ctor(Muc_mod$Dose) head(Muc_mod) #view firs few rows of data summary(Muc_mod) sum_data3 = ddply(Muc_mod, c("Group", "Dose"), summarise, N = sum(!is.na(Rrs)), mean = mean(Rrs, na.rm=TRUE), sd = sd(R rs, na.rm=TRUE), se = sd / sqrt(N)) boxplot(sum_data3) dev.new() plot(Muc_mod$Dose, Muc_mod$Rrs, data(Muc_mod), colour = Muc_mod$Group) # Use position_dodge to move overlapped errorbars horizontally dev.new() ggplot(sum_data3, aes(x=Dos e, y=mean, group=Group, color=Group)) + geom_errorbar(aes(ymin=mean se, ymax=mean+se), width=.1, position=position_dodge(0.05)) + geom_line() + geom_point() + scale_colour_manual(values = c("#650EB0", "#DD035B", "chocolate 1","#67BB 00")) + labs(title="Airway Hyperreactivity",x= expression(paste("MCh ("*mu*"g/kg/min, iv)")),

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90 y = expression(paste("Airway Resistance (", cmH[2]*O~"/ml/s"^" 1"*")"))) + theme(plot.title = element_text(hjust = 0.5)) + scale_y_continuous( limits=c(0, 32.5)) ####### Power Tests ##### challenged = first$Rrs sd(challenged, na.rm = TRUE) power.t.test(n = 13, 0.2, sd = 10.62247 power = NULL) ###***********************##### #Perform ANOVA to get F value and P value. fit_1 = aov(Rrs ~ Group, dat a = Muc_mod) summary(fit_1) #Try some comparisons with the multicomp package. fit_mc = glht(fit_1, linfct = mcp(Group = "Dunnett"), alternative = "less") summary(fit_mc, test = adjusted(type = "single step")) fit_mc = glht(fit_1, linfct = mcp (Group = "Dunnett"), p.adjust.methods = "none") summary(fit_mc)