WO2012170084A1 - Composition and method for treating lung disease and mucous cell metaplasia - Google Patents

Abstract

Embodiments of the present invention relate to biologically and pharmacologically active Bik Peptides for use as therapeutic agents for the treatment of lung disease, pulmonary disease and chronic lung disease, and mucous cell metaplasia (MCM) in airways of the lung. They also relate to methods of modulating Bik in airway epithelial cells (AECs) and methods of treating mucous cell metaplasia.

Description

COMPOSITION AND METHOD FOR TREATING LUNG DISEASE AND MUCOUS CELL

METAPLASIA CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of the filing of U.S. Provisional

Patent Application Serial No. 61/444,957, entitled "COMPOSITION AND METHOD FOR TREATFNG LUNG DISEASE AND MUCUS CELL METAPLASIA", filed on February 21, 2011, and the specification and claims thereof are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with government support under Grant No. ROl HL068111 awarded by the NIH. The government has certain rights in the invention.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT

DISC

Not Applicable.

COPYRIGHTED MATERIAL

[0004] Not Applicable.

INTRODUCTION

[0005] The embodiments of the present invention relate generally to the field of lung disease, pulmonary disease and chronic lung disease, mucous cell metaplasia (MCM) in airways of the lung, methods of modulating Bik in airway epithelial cells (AECs) and methods of treating mucous cell metaplasia and mucous hypersecretion. [0006] MCM with mucous hypersecretion is a prominent manifestation of many common inflammatory diseases of the upper and or lower airways. Excess production of mucus is a problem in both minor and serious illnesses, ranging from the common cold to fatal exacerbations of chronic bronchitis, asthma, and cystic fibrosis for example. Control of mucous hypersecretion is a difficult challenge because of the complex roles of mucus in host defenses, the variety of stimuli that can induce mucous secretions, the redundancy of the signal transduction pathways involved, and the multiplicity of mucin genes.

Available treatments for airway diseases, including anti-inflammatory drugs, antibiotics, and bronchodilators, are generally ineffective for suppression of mucous secretions .

[0007] One of the major hallmarks of airway remodeling in asthma or other diseases of the lung is the appearance of mucous cells in airways that are normally devoid of these cells. This condition is called MCM. In the lung, airway epithelial remodeling can be life threatening, since MCM is the foundation for hypersecretion that can obstruct the airway lumen. MCM poses a serious risk of reducing airflow in the lung due to acutely secreted mucus that can sometimes completely obstruct the airways and lead to death of asthmatics for example. In addition, epidemiological studies suggest that patients with chronic MCM are at a higher risk of developing lung cancer. Therefore, reducing hyperplastic cells in patients with chronic MCM, such as chronic bronchitics, may reduce their risk of developing lung cancer.

[0008] Cigarette smoking is the leading cause of disease for 25-27 million individuals with chronic obstructive pulmonary disease (COPD) in the United States alone and for over 350 million people world-wide. Cigarette smoke alone or in combination with environmental pollutants or allergens can induce an inflammatory response in the lungs of individuals. With 1.25 billion people smoking cigarettes daily worldwide, these diseases are expected to reach epidemic proportions in the next decade. In humans, goblet cell metaplasia is a frequent finding in the large and small airways of cigarette smokers, as are increases in the number and size of mucus-producing glands in the large airways. Indeed, the amount of intraluminal mucus in the small airways is responsible for reduced lung function and represents a major difference between subjects with moderate to severe COPD (GOLD stages 3 and 4) and those with a lesser degree of airflow obstruction (GOLD stages 1 and 2). In addition, excess mucous secretions in the airways are important in the pathogenesis of acute exacerbations of COPD.

[0009] Contact of the airway epithelium with bacteria or viral infectious agents and allergens (environmental pollutants) elicits an inflammatory response that recruits polymorphonuclear cells and macrophages to the airways and initiates the proliferation of epithelial cells. The number of epithelial cells that produce sufficient mucins and protection from further injury is increased by proliferation, but when sustained for longer periods can be the basis for airflow obstruction in subjects with asthma and chronic bronchitis.

[0010] IFNy and STAT1 signaling are required for the resolution of allergen-induced

MCM during prolonged exposure to allergen. IFNy induces cell death in only

proliferating AECs regardless of whether mucins are expressed. However, because mucous cells appear to be the cells that become hyperplastic following inflammatory responses, this cell type is targeted by IFNy in vivo. Efforts to identify which cell death regulatory protein(s) IFNy may need to induce cell death in airway epithelial cells unexpectedly led us to discover that Bik is necessary for IFNy to cause cell death in hyperplastic epithelial cells.

[0011] Thus, what is needed is a composition and method for modulating proliferating

AEC's and/or MCM and or methods, compositions for modulating Bik as a therapeutic treatment for patients in need thereof.

SUMMARY OF EMBODIMENTS OF THE INVENTION

[0012] It is an object of the embodiments of the present invention to modulate Bik

activity in vivo. [0013] It is another object of the embodiments of the present invention to modulate Bik activity in vitro.

[0014] It is yet another object of the embodiments of the present invention to treat MCM for example in the airways of the respiratory system.

[0015] It is still another object of the embodiments of the present invention to monitor

Bik protein levels to determine the health of an individual with and without airway disease.

[0016] It is a further object of the embodiments of the present invention to provide a therapeutic amount of Bik polypeptide to a subject in need thereof in vivo.

[0017] It is an additional object of the embodiments of the present invention to provide a

Bik peptide to a target cell in vitro.

[0018] It is another object of the embodiments of the present invention to treat lung disease.

[0019] It is another object of the embodiments of the present invention to monitor Bik protein as a biomarker for the health of a patient.

[0020] It is another object of the embodiments of the present invention to monitor Bik gene expression as a biomarker for the health of a patient.

[0021] It is another object of the embodiments of the present invention to monitor Bik protein and or gene expression as a biomarker in individuals with lung disease and/or those receiving treatment for lung disease.

[0022] Additional objects and advantages of the embodiments of the present invention will be apparent in the following detailed description read in conjunction with the accompanying drawing figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

[0024] Fig. 1 illustrates that Exposure to CS decreases Bik expression;

[0025] Fig. 2 illustrates that Exposure to CS leads to increased epithelial cell hyperplasia

(ECH).

[0026] Fig. 3 illustrates that Restoration of Bik Expression Reduces CS-induced MCM and ECH;

[0027] Fig. 4 illustrates that Bik expression reduces nuclear localization of CS-activated

ER l/2 and phopho-ERKl/2 enhances Bik-induced cell death;

[0028] Fig. 5 illustrates that Bik causes cell death by interacting with DAPK to inhibit nuclear localization of activated ERKl/2;

[0029] Fig. 6 illustrates that Activated Bak is part of the Bik/phospho-ERK/DAPK

complex during the cell death-inducing process;

[0030] Fig. 7 illustrates a Proposed IFNy-induced pathway that results in killing of

proliferating NHBECs and how cigarette smoke suppresses Bik expression and blocks the killing of NHBECs;

[0031] Fig. 8 illustrates that Peptide SEQ ID NO. 1 is a moderate killer of primary

epithelial cells compared to Peptide SEQ ID NO. 2; [0032] Fig. 9 illustrates that Peptide SEQ ID NO. 17 is the most active peptide to cause cell death in cells with low Bcl-2 levels in comparison to Peptides SEQ ID NO. 1, 2, 16, and 18.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The intrinsic apoptotic pathway that is responsible for removing excess numbers of mucous cells may be dysfunctional in individuals with asthma and cystic fibrosis. In an effort to determine which of the Bcl-2 family of proteins plays a role in sustaining MCM in chronic bronchitis as an example of allergen induced lung disease, we screened for the expression of all Bcl-2 family mRNAs in airway epithelial cells (AECs) from patients with chronic bronchitis and controls without lung diseases. The cell death of AECs during the resolution of metaplastic mucous cells is regulated by the Bcl-2 family of proteins, and involves the intrinsic apoptotic pathway.

[0034] During the resolution of allergen-induced epithelial and mucous cell hyperplasia that is mediated by IFNy, STAT1 and Bik, (a Bcl-2 family member), participate in clearing cell hyperplasia because unlike wild-type mice STAT1- and Bik-deficient mice fail to resolve MCM. Consistent with these findings, mouse airway epithelial cells (MAECs) from STAT1- and Bik-deficient mice are resistant to IFNy-induced cell death. Following inflammatory responses to LPS, allergen or cigarette smoke, up to 30% of hyperplastic cells undergo death to eliminate excess mucous cells and to return to the original cell numbers. Disruption of this recovery process may lead to persistent elevation of mucous cell numbers and contribute to chronic mucous hypersecretion and airway obstruction found in chronic lung diseases such as cystic fibrosis, asthma, and chronic bronchitis. The cell death of AECs during the resolution of metaplastic mucous cells is regulated by the Bcl-2 family of proteins and involves the intrinsic apoptotic pathway.

[0035] Bronchial brushings were obtained from 11 subjects each with chronic bronchitis, and 9 controls with no evidence of lung disease (Table 1). Screening for dysregulated expression of Bcl-2 family of mRNAs identified Bik levels to be significantly reduced in bronchial brushings of patients with chronic bronchitis compared to non-diseased controls as is illustrated in figure 1A. Referring now to figure 1, exposure to cigarette smoke reduces Bik expression. In figure 1 Bar=Means ± standard error from the mean (n=4 mice/group or n=3 different treatments/group). * denotes statistically significant difference (P < 0.05). Box plots represent the median value and 25th and 75th quartile. Whiskers are the 5th and 95th percentile. Open circles are outlier values (> 1.5 x interquartile range). Results for Bnip32, Bok, Bid showed no significant differences among groups. A standard linear model applied to the quantitative RT-PCR data and F- statistics and p-values calculated from the linear fit showed that Bik was significantly reduced in bronchial brushings of chronic bronchitics compared to non-diseased controls (p < 0.05). Levels of mRNAs encoding for Bnip3L, Bok, Bid (figure 1A), and Bcl-2, Mcl-1, Puma (data not shown) were not different among these groups. This finding was confirmed by analyzing lung tissues obtained at autopsy from current and never smokers with and without chronic bronchitis respectively.

[0036] Reduced Bik but increased MUC5AC mRNA levels were also detected when normal human airway epithelial cells (HAECs) were exposed to cigarette smoke (CS), or when autopsy tissues from former smokers with and without chronic bronchitis were compared figure IB (NS-NCB=never smokers without CB; CurS-CB=current smokers with CB) and figure IC (Bik and MUC5AC mRNAs levels in the lungs of C57BL/6 mice exposed to 250 mg/m3 CS or filtered air for 6 h/d, 5 d/wk for 10 wks. Bik mRNA).

Similarly, exposure of C57B1/6 mice to CS resulted in increased number of epithelial and mucous cells per mm basal lamina along with reduced Bik but increased Muc5ac expression figure ID. This change was sustained even when mice were allowed to recover in filtered air for 8 weeks.

[0037] Referring now to figure IB, MUC5AC mRNA levels were increased in the lung tissues of current smokers with chronic bronchitis compared to controls. This observation was also confirmed in C57BL/6 mice that were exposed to 250 mg/m3 of mainstream CS or filtered air for 6 hours/day, 5 days/week for 10 weeks as is illustrated in figure IC. Bik mRNA levels were reduced by 50% and the Muc5ac levels increased ~3-fold in the lungs of CS-exposed mice compared to controls. IFNy induces Bik expression in AECs within 24 hours of treatment (data not shown). Referring now to figure ID and IE (levels in HAECs treated with nothing as control or 1000 ng/ml CSE for 24 h and with 50 ng/ml IFNy for the following 24 h), while IFNy induced Bik expression in HAECs, CS treatment suppressed this effect both at the mRNA and protein levels. Collectively, these findings demonstrate that CS is a potent suppressor of Bik expression both in airway epithelia of patients with chronic bronchitis and in mouse lungs, even under conditions when a pro- apoptotic stimulus like IFNy is present.

100381 Bik expression was not only suppressed in the biopsy and autopsy lung tissues of cigarette smokers compared to non-smokers, but also in lung tissues of former smokers with chronic bronchitis compared to former smokers without. These findings suggest that Bik levels are not only reduced by cigarette smoking, but the level of reduction is driven by factors that may be directly associated with susceptibility to developing chronic bronchitis. It appears that Bik is suppressed by CS more drastically in former smokers with chronic bronchitis and this reduction allows increased number of mucous cells to be sustained compared to former smokers without chronic bronchitis. It is possible that airway cells in people who are genetically predisposed to chronic bronchitis either have reduced baseline Bik lev els or once exposed to CS, permanently and more efficiently degrade Bik mRNA compared to smokers without chronic bronchitis. Therefore, Bik is a useful biomarker that predicts susceptibil ity to chronic bronchitis. Factors that differentially regulate Bik expression in subjects with chronic bronchitis will aid in management of individuals who may hav e chronic bronchitis and those that were former smokers.

[0039] Interestingly, suppression of Bik expression was associated with significant

increases in MUC5AC mRNA levels both in human primary HAECs and in the lungs of mice exposed to CS. I n mice, these changes were also associated with an increased number of epithelial cells per mm basal lamina compared to those exposed to filtered air as control. These findings suggest that the reduction of this pro-apoptotic protein allows sustained presence of hyperplastic epithelial cells that synthesize and secrete mucus. [0040] Ectopic expression of Bik using adenoviral vector reversed CS-induced MCM by forming a complex blocking nuclear translocation of activated ERK1/2 to mediate cell death in AECs. Hence, targeted expression of Bik may be useful to control CS-induced MCM and help reduce symptoms associated with excessive mucous secretion in chronic pulmonary diseases.

[0041] Referring now to figure IF, qRT-PCR analysis of Bik mRNA transcripts after terminating cellular mRNA synthesis with 5,6-dichloro-l-B-D- ribofuranosylbenzimidazole (DRB) was examined to determine the time-dependent decay of Bik mRNA. CS reduces Bik mRNA half-life from 3.1 to 1.4 hours. HAECs were treated with 50 ng/ml transcription inhibitor 5,6-dichloro-l- B-D- ribofuranosylbenzimidazole (DRB) in the presence and absence of CS over a period of 0, 1, 2 and 4 hours. The time-dependent decrease of Bik in RNA was examined using qRT- PCR. These results suggest that CS reduced Bik expression by destabilizing Bik mRNA.

[0042] In figure 2 Bars=group means ± standard error from the mean (n=4 mice/group or n=3 different treatments/group). * denotes significant differences among groups (P < 0.05). Referring now to figures 2A 2B, shown are airway epithelial cell numbers in bik+/+ and bik-/- mice exposed to 250 mg/m3 CS or filtered air for 6 h/d, 5 d/wk for 10 wks (A), or 3 wks followed by 8 wks of recovery in filtered air (B). The left lungs were fixed under constant pressure perfusion, cut into 4-mm slices from distal to caudal and slices embedded in paraffin. Tissue sections (5 um) were stained with hematoxylin and eosin and the total epithelial cell number in the airways quantified. In figure 2A, the number of AECs per mm of basal lamina was significantly higher in the lung tissues of both CS-exposed bik+/+ and bik-/- mice compared to the filtered air-exposed control groups. To investigate whether Bik remains suppressed even after cessation of CS, we examined whether the resolution of CS-induced ECH is affected when bik+/+ and bik-/- mice are exposed to CS or filtered air for 3 wks and allowed to recover in filtered air for 60 days. In these mice, the neutrophilic inflammatory response was resolved and was not different from air-exposed controls (data not shown). Referring now to figure 2B, the increase in epithelial cell number was sustained both in bik+/+ and bik-/- mice exposed to CS after 60 days in filtered air. Furthermore, the reduction in Bik induced Bik expression in a STATl -dependent manner. However, treatment of HAECs with CS did not reduce STATl activation (data not shown), suggesting that CS-induced reduction of Bik expression occurs independently of the STATl pathway. Furthermore, in HI 299 or HEK- 293 cells CS did not affect Luciferase activity of a 2kb Bik promoter construct that was inserted into the pGL3- Basic firefly luciferase reporter plasmid (Promega, Madison, WI) (data not shown). The reduction of Bik mR A and increase in Muc5ac levels were sustained in bik+/+ C57BL/6 mice that were exposed to 250 mg/rri3 CS or filtered air for 6 h/d, 5 d/wk for 3 wks followed by 8 wks of recovery in air as is illustrated in figure 2C. Bik and Muc5ac mR As quantified by qRT-PCR. Bik is suppressed in former cigarette smokers that have persistent chronic bronchitis. Therefore, we compared lung tissues from former smokers with and without chronic bronchitis (Table 2). Referring to figure 2D, Bik mRNA levels were reduced, whereas MUC5 AC mRNA levels were increased significantly in the lung tissues of former smokers with chronic bronchitis compared to those without (quantified by qRT- PCR. (FS-NCB=former smokers without CB; FS-CB=former smokers with CB)).

Referring now to figure 2E, to further investigate whether Bik is suppressed by CS, primary HAECs from 5 individuals differentiated in an air-liquid interface (ALI) culture were treated with a CS extract that contained 0 or 1000 ng/ml total particulate matter for 24 ours and harvested 5 days later. Bik and MUC5AC mRNA levels were quantified from CS-treated and nontreated controls by qRT-PCR. Similar to what was observed in patients with chronic bronchitis Bik mRNA levels were reduced 3-fold in CS-treated cultures compared to non-treated controls. In these CS-treated NHBECs MUC5AC mRNA levels were significantly increased. Mucus cell numbers per mm basal lamina were increased compared to nontreated controls as is illustrated in figure 2F, in which Primary HAECs placed in culture in an ALI culture were left untreated or treated with 1000 ng/ml CS for 24 h and 3 d later membranes were embedded in paraffin and stained with AB/PAS. The number of mucous cells was significantly increased in CS-treated culture compared to untreated controls. Representative photomicrographs of AB/PAS stained culture showing increased number of mucous cells in primary differentiated HAECs treated with 1000 ng/ml CSE for 24 h and harvested 3 d later, compared to untreated groups. These findings suggest that in mice CS exposure suppresses Bik expression in a permanent manner. A similar mechanism may cause increased mucous hypersecretion in former smokers with chronic bronchitis as the resolution of CS-induced ECH and MCM was disrupted. Because Bik levels were reduced in patients with chronic bronchitis and in mice exposed to CS, we explored whether ectopic expression of Bik would reduce CS-induced increase in ECH and MCM. Referring now to figure 3, treated primary HAECs that were differentiated in an air-liquid interface (ALI) culture with 1000 ng/ml CS extract for 24 hours were analyzed. The cultures were infected with nothing, Ad-Bik, or Ad-BikL61G as control. BikL61G is a mutant Bik with the conserved Leu residues in the BH3 domain substituted by Gly. Immunostaining with MUC5 AC antibodies showed that Ad-Bik significantly reduced the number of mucous cells in the differentiated HAEC cultures compared to non- or BikL61G-infected cultures as is illustrated in figure 3 A. Similar results were obtained for primary HAECs by Alcian blue and periodic acid Schiff (AB/PAS) staining (data not shown). To investigate whether the expression of Bik would reduce CS-induced ECH in vivo, we exposed mice to 250 mg/m3 CS or filtered air for 6 h/d, 5 d/wk for 3 wks, and intranasally instilled them with 109 pfu HA-tagged Ad-Bik or Ad-GFP or PBS as control on two consecutive days following exposures. Mice were sacrificed one day later and lungs from HA-Ad-Bik- and Ad-GFP-instilled mice were analyzed by Western blotting. The HA-tagged Bik was detected in mice instilled with Ad-Bik but not in those instilled with Ad-GFP as is illustrated in figure 3B. In addition, CS-induced ECH was significantly reduced in the airways of mice instilled with Ad-Bik compared to those instilled with Ad-GFP or PBS as is illustrated in figure 3C.

Furthermore, immunofluorescence staining showed that Muc5ac positivity was reduced in CS-exposed mice instilled with Ad-Bik compared to those instilled with Ad-GFP or PBS as is illustrated in figure 3D. Bars=group means ± SEM (n=6 mice/group). * denotes significantly different from Ad-Bik (P < 0.05). These findings signify that targeted restoration of Bik expression is useful to control CS-induced ECH and MCM. [0045] The fact that CS-induced MCM and ECH in differentiated primary HA EC cultures and in vivo mice were reduced when Bik expression was restored suggests that Bik must be inducing death of mu cm-producing cells. Previous studies showed that proliferating basal cells give rise to a subpopulation of mucous cells that retain the ability to div ide while others lose this proliferative phenotypc and become fully differentiated. Therefore, susceptibility of only proli ferating A EC's to death signal s may ensure that only hyperplastic A EC's are removed during the resolution process without damaging the barrier function of the airway epithelium. Hyperplastic cells in airways appear to be primarily mucus-producing cells. Hence, restoring Bik expression or elevating portion of Bik protein that induces cell death of AEC may reduce hyperplastic epithelial cells, thereby reducing MCM. This selective targeting of hyperplastic cells restores the normal proportions of cell types in airways without destroying the integral barrier function and innate protective mechanism of the airway epithelium.

[0046] Restoring Bik expression using an adenoviral expression system significantly suppressed CS-induced increase in epithelial and mucous cells in differentiated HAEC cultures and in airways of mice. Bik interacted with and blocked nuclear translocation of phospho-ER l/2 and caused cleavage of the death-associated protein kinase (DAPK) to induce death of HAECs. These studies show that restoring Bik expression using appropriate vectors may be a therapeutic approach for reducing mucous hypersecretion in chronic bronchitis. In addition it is shown that following inflammatory responses to LPS or allergen, up to 30% of hyperplastic cells undergo death to eliminate excess mucous cells and to return to the original cell numbers. Disruption of this recovery process may lead to persistent elevation of mucous cell numbers and contribute to chronic mucous hypersecretion and airway obstruction found in chronic lung diseases such as cystic fibrosis, asthma, and chronic bronchitis.

[0047] In figure 4, Bars=group means ± standard error from the mean (n=3 different treatments/group). * denotes statistically significant difference (P < 0.05). Referring now to figure 4A, protein lysates analyzed for activated ERKl/2 by Western blotting showed that in HAECs, ERKl/2 was activated within 5 minutes of 1000 ng/ml CSE treatment and this activation was sustained for 15 minutes. HAECs cells were infected with 100 MOI Ad-Bik or Ad-BikL61G and 24 hours later treated with CS extract for 15 minutes.

Analysis of cytosolic and nuclear fractions showed that activated ERKl/2 was reduced in the nuclear fraction of Ad-Bik- compared to Ad-BikL61G -expressing cells which is illustrated in figure 4B. Interestingly, CS enhanced Ad-Bik-induced cell death, while Ad- BikL61G had little effect on CS-treated HAECs as is illustrated in figure 4C, where HAECs infected with Ad-Bik or Ad-BikL61G at 100 MOI were either treated with CSE or left untreated and counted 24 h later. These results support a cell death that is enhanced when activated ERKl/2 is retained in the cytosol by Ad-Bik and that the conserved Leu residue within the BH3 domain of Bik is important for the inhibition of translocation. Retention of activated ERKl/2 enhances Ad-Bik-induced cell death. HAECs were treated with another stimulus that is known to activate ERKl/2. HAECs were treated with different concentrations of IGF- 1 for 0-30 min and protein lysates analyzed for p-ERKl/2, ERKl/2 and actin by Western blotting. ERKl/2 was activated earlier and with increasing intensity when cells were treated with increasing IGF-1 concentrations as is illustrated in figure 4D. HAECs were infected with 100 MOI Ad-Bik or Ad-BikL6io and treated with different concentrations of IGF- 1. Cell viability was quantified by trypan blue exclusion assay. IGF-1 enhanced Ad-Bik-induced cell death in a dose-dependent manner as is illustrated in figure 4E. HAECs were treated with 1 μΜ ERKl/2 inhibitor, U0126, which effectively reduced the levels of activated ERKl/2 as detected by Western blotting as is illustrated in figure 4F. Inhibition of ERKl/2 activation using U0126 suppressed Ad-Bik-induced cell death, while U0126 alone had no effect on growth of HAECs cells as is illustrated in figure 4G, where HAECs were treated with 1 μΜ U0126 and infected with 100 MOI Ad-Bik followed by 1000 ng/ml CSE treatment 24 h later. Cell death induced by Ad-Bik in the presence of CS was diminished significantly when ERKl/2 activation was inhibited by 1 uM U0126. In figure 4, Bars=group means ± standard error from the mean (n=3 different treatments/group). * denotes statistically significant difference (P < 0.05). Together, these findings illustrate that Bik retains activated ERKl/2 in the cytosol to cause cell death. [0049] Bik-induced cell death was enhanced by CS- or IGF- 1 -induced ERKl/2 activation. Blocking ERKl/2 activation using U0126 suprisingly allev iated Bik-induced cell death suggesting that ERK l /2 activation may be an integral functional part of Bik- induced cell death. The fact that the presence of serum was requi ed for Bik to inhibit nuclear localization of phospho-ERKl/2 in H 1975 cells and that the 60 kDa DAPK cleavage product showed a stronger interaction with the Bik-ERK 1/2 complex suggests that DAPK is essential for cytosolic retention of phospho-ERKl/2. ERK activation has generally been associated with cell survival and proliferation; however, a number of studies show that depending on the stimuli and cell types involved, activation of ERK can mediate cell death (reviewed by Mebratu). Activation of ERKl/2 stimulates apoptosis in cells expressing DAPK and serum-induced phosphorylation of DAPK by ERK enhancing its kinase activity and death promoting effects. Bik is a link between ERKl/2 and DAPK to lead toward death pathways.

[0050] Activation of EGFR immediately and efficiently activates ERKl/2. Cancer cells that have strong activation of ERKl/2 due to persistent activation of EGFR would be highly susceptible to Ad-Bik. Referring now to figure 5, Bik is linked to cell death by interacting with DAPK to inhibit nuclear localization of activated ERKl/2. Lung adenocarcinoma cell lines that have wild-type EGFR (HI 299) or mutations in EGFR (H1975) were selected. Both H1299 and H1975 cells showed increased activated ERKl/2 when cultured in serum-containing medium as is illustrated in figure 5A. The EGFR mutant cell line, HI 975, was more sensitive to Ad-Bik-induced cell death at the lower MOI of 1 and 10 as is illustrated in figures 5B and 5C (cell viability 24 h after infection with 0, 1, 10, or 100 MOI Ad-Bik quantified by Trypan blue exclusion). Bars=group means ± standard error from the mean (n=3 different treatments/group). * denotes statistically significant difference. HI 975 was selected for further study as to the mechanism by which Ad-Bik induces cell death. Bik overexpression inhibited nuclear localization of activated ERKl/2 in serum-treated but not in serum- starved HI 975 cells as is illustrated in figure 5D, where Nuclear and cytosolic protein preparations 24 h after infection of HI 975 cells with 100 MOI Ad-Bik or Ad-BikL61G were analyzed for phospho-ERKl/2, total ERKl/2, Bik, lamin, and actin, by Western blotting. Referring now to figure 5E, immunoprecipitation assays with proteins extracted from HI 975 cells that were infected with Ad-Bik, or Ad-BikL61G were performed. The cell lysates (input) and immunoprecipitates were resolved by SDS-PAGE and probed by Western blotting using antibodies to Bik, phospho-ERKl/2, total ERKl/2. Increased phospho-ERKl/2 was detected in pull-down products from Ad-Bik compared to Ad-BikL61G-infected cells despite increased levels of Ad-BikL61G being present as is illustrated in figure 5E.

[0051] Ad-Bik was over expressed in HI 975 cells and protein lysates were

immunoprecipitated using anti-p-ERKl/2 antibody. The lysates were examined for the presence of DAPK by Western blotting. Both Bik and phospho-ERKl/2 were present in the pull-down products. In addition to the 160 kDa DAPK protein, two additional 100 and 60 kDa proteins were detected with the DAPK antibodies. Interestingly, only the 160 and 100 kDa DAPK proteins were detected in the input but not the 60 kDa DAPK protein as is illustrated in figure 5F.

[0052] The 60 kDa DAPK protein is a DAPK breakdown product that interacted with

ERKl/2 and Bik and contributes to clearage of DAPK. The 160 kDa DAPK protein levels in HI 975 cells expressing Ad-Bik or Ad-BikL61G were compared. It was found that DAPK levels were reduced in cells expressing wild-type compared to mutant Bik as is illustrated in figure 5G, where Protein lysates from non-treated HI 975 cells or cells infected with Ad-Bik or Ad-BikL61G for 24 h were resolved by SDS-PAGE and probed with antibodies to DAPK, Bik and actin. bik+/+ and bik-/- MAECs were treated with 1000 ng/ml CS extract for 4 or 8 hours. As is illustrated in figure 5H, DAPK levels were not affected at 4 hours but were reduced in bik+/+ but not in bik-/- MAECs 8 hours after CS treatment. Similarly, DAPK levels were also reduced in bik+/+ but not in bik-/- MAECs that were incubated in serum-containing medium for 24 hours; conditions that favor activation of ERKl/2 as is illustrated in figure 51. These findings support the hypothesis that activated ERKl/2 in the presence of Bik facilitates cleavage of DAPK. [0053] Referring now to figure 5J, immunoprecipitation of protein lysates with anti-p-

ER l/2 antibodies from cells maintained with or without serum and infected with Ad-Bik showed that Bik and the 60 kDa DAPK product were detected more in cells cultured with serum compared to those without. Similar to the previous finding, the 100 but not the 60 kDa DAPK protein was detected in the input. These findings suggest that activation of ERKl/2 in the presence of Bik may lead to the cleavage and formation of a protein complex with the 60 kDa DAPK that sequesters ERKl/2 in the cytosol and facilitates cell death.

[0054] The role of DAPK in Bik-induced cell death was confirmed by suppressing

DAPK expression using a retroviral vector expressing short hairpin RNA (shRNA). Ad- Bik-induced cell death was significantly reduced in H 1975 cells with suppressed DAPK compared to those infected with control retrovirus as is illustrated in figure 5K.

10055| In H 1975 cells expressing Bik, three distinct bands with molecular weights of approximately 160, 100, and 60 kDa were detected using the anti-DAPK antibodies. Interestingly, while all three forms were detected in the pull-down products using anti-p- ERKl/2 antibodies, only the 100- and 160-kDa products were detected in the input.

These findings suggest that during the Bik-induced cell death process, the 160 DAPK full- length protein is cleaved to form the smaller molecular weight proteins.

[0056] The BH3-only domain proteins of the Bcl-2 family transduce multiple death

signals to the mitochondrion or ER and interact with anti-apoptotic Bcl-2 family members to trigger Bax/Bak mediated cell death. The peptides that constitute the BH3 domain bind to the minor groove of the anti-apoptotic proteins Bcl-2 and Bcl-xL to inactivate their anti-apoptotic function, or they can activate pro-apoptotic genes, such as Bax and Bak. Peptides that encompass the BH3 domain linked to the TAT protein, tagged with polyarginine peptide or myristylated to facilitate entry into NHBECs or hydrocarbon staples to ensure stability were designed to modulate Bik activity. Our results indicate that hydrocarbon stapled peptides enter MTECs and NHBECs. These peptides are also expressed using adenoviral vectors. Mutation of the Leu residue within the BH3 domain abrogates the pro-apoptotic function of Bik.

[0057] Peptides that comprised Be 1-2 homology region 3 are illustrated below and in the

Sequence Listing filed with this application. It is noted that other peptides containing at least four contiguous amino acids of human Bik BH3 domain or a variant thereof or a homologue of human Bik BH3 domain or a variant thereof can potentially have the same effect as the complete human Bik protein when reintroduced into cells. Thus, peptides with sequences that are adjacent to the sequences of the peptides illustrated below have potential for further studies. In order to enable hydrocarbon staple for stabilization and to facilitate entry into cells, in the sequences below, some of the amino acids are modified to norleucine (Nle), and (S)-2(2'-pentenyl) Ala can optionally be used at the sites denoted by the letter X. However, any amino acid may be substituted at these locations for use with other techniques to facilitate entry into cells. 5-FAM is a green fluorescent reagent used to label peptides. Certain peptides contain special cyclization to get double bond.

[0058] Stapled Peptide Sequence 5-FAM-EGSDALALRLAXIGDX-Nle-DVSLRA (SEQ

ID NO: 1);

[0059] Scrambled peptide 5-FAM-ARLIGESVADSXDALX-NLe-LARDGL (SEQ ID

NO: 2).

[0060] Generally, death increased in HI 299 cancer cells and airway epithelial cells

treated with Peptides SEQ ID NO. 1 and 2 after 72-96 hours. Referring to figure 8, Peptides SEQ ID NO. 1 and 2 were compared in their ability to cause death in primary airway epithelial cells. The results showed a higher reduction in cell number with the Peptide SEQ ID. NO. 1 treatment.

[0061] When primary HAEs were differentiated on Transwell membranes and treated with scrambled peptide SEQ ID NO. 2 the amount of mucus that was harvested from the top of the cultures was approximately 20 microliter, while the ones treated with Bik peptide showed no mucus.

[0062] 5-FAM-EGSDALALRGAXIGDX-Nle-DVSLRA (cyclic) (SEQ ID NO: 3);

[0063] 5-FAM-ALALRLAXIGDX-Nle-DVSLRA (SEQ ID NO: 4);

[0064] 5-FAM-NNNNLAXNNNNX-NLe-NNNNNN (N = any amino acid) (SEQ ID NO: 5);

[0065] 5-FAM-ALALRLAXIGDX-Nle-DV (SEQ ID NO: 6);

[0066] 5-FAM-RLAXIGDX-Nle-DVSLRA (SEQ ID NO: 7);

[0067] 5 -F AM-RL AXIGDX-Nle-D V (SEQ ID NO: 8);

[0068] 5-FAM-LAXIGDX-Nle-D(SEQ ID NO: 9);

[0069] EGSDALARLAXIGDX-Nle-DVSLRA(SEQ ID NO: 10);

[0070] ATLAXIGDX-Nle-DVSLRA (SEQ ID NO: 11);

[0071] ALARLAXIGX-Nle-DV (SEQ ID NO: 12);

[0072] RLAXIGDX-Nle-DVSLRA (SEQ ID NO: 13);

[0073] RLAXIGDX-Nle-DV (SEQ ID NO: 14);

[0074] LAXIGDX-Nle-D (SEQ ID NO: 15);

[0075] 5-FAM-EGSDALALRLASIGDX-Nle-DVXLRAK (SEQ ID NO. 16);

[0076] 5-FAM-EGSXALAXRLASIGDE-Nle-DVSLRAK (SEQ ID NO. 17); [0077] 5-FAM-EGSXALAXRLASIGDX-Nle-DVXLRAK (SEQ ID NO. 18).

[0078] Referring now to figure 9, Peptides SEQ ID NO. 1, 2, 16, 17, and 18 were used to treat primary airway epithelial cells from 5 individuals that have different levels of Bcl-2, an anti-apoptotic protein that inhibits Bik-induced cell death. Persons 1 and 2 have low and persons 3-5 have higher levels of Bcl-2. The cells were cultured on 12-well dishes using BGEM medium as described previously. Cells were then treated with 5 micromolar of peptide and maintained in culture for 3 days. Cells were then harvested and cell counts determined for each individual. Results show that all peptides were effective in reducing cell numbers in the samples with low Bcl-2 levels. Peptide SEQ ID NO. 17 was the most effective in reducing the number to below 50%. For the persons with higher levels of Bcl- 2 only peptide SEQ ID NO. 17 showed an effective reduction in cells numbers. The other two samples with higher levels of Bcl-2 were unaffected by these peptides.

[0079] Referring now to figure 6, Bak can be activated to cause cell death following the interaction of Bik, phosphor-ER l/2, and DAPK. DAPK expression was knocked down in HI 975 cells through stable expression of short hairpin DAPK (shDAPK). HI 975 cells expressing shDAPK and cells expressing a control hairpin (shCon) were infected with 100 MOI Ad-Bik expressing Bik tagged with the HA epitope. Cell lysates were

immunoprecipitated with anti-HA to bind the tagged Bik and with phospho-ERK antibodies. The cell lysates (input) and immunoprecipitates were resolved by SDS-PAGE. Referring to figure 9(A), Western blot analysis with antibodies to activated Bak (Ab-1 antibody), total Bak, phosphor-ERKl/2, DAPK, and HA showed that the tagged Bik interacted with activated. The effects of Peptide SEQ ID NO. 17 on the Bak and DAP- kinase processes may operate by activating the same machinery as the complete Bik protein.

[0080] BIK mimetic therapies can be formulated in a pharmaceutical composition for administration to a mammalian patient. As used herein, a "pharmaceutical composition" includes the active agent and a pharmaceutically acceptable carrier, excipient or diluent. [0081] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce a severe allergic or similar untoward reaction when administered to a mammal. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particular in humans.

[0082] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or other aqueous solutions, saline solutions, aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

[0083] For human therapy, the pharmaceutical compositions, including the active agents, will be prepared in accordance with good manufacturing process (GIMP) standards as set by the Food & Drug Administration (FDA). Quality assurance (QA) and quality control (QC) standards will include testing for purity and function, homogeneity and function, and/or other standard measures.

[0084] In order to treat an allergen-mediated or CS mediated lung disease and/or its symptoms, the pharmaceutical composition hereof is administered by any route that will permit delivery of the active agent to the affected cells. Since it is believed that Bik polypeptide or mimetic therapy does not harm normal cells, systemic administration of the active agent is acceptable. Preferably, administration is intraperitoneal and also including, but not limited to, inhalation, intra-arteriole, intramuscular, intradermal, subcutaneous, perenteral, intraventricular, and intracranial administration. Alternatively, the active agent may be delivered locally to the affected cells by any suitable means.

[0085] In therapeutic treatments of the invention, a therapeutically effective amount of the pharmaceutical composition is administered to a mammalian patient. As used herein, the term "therapeutically effective amount" means an amount sufficient to reduce by at least about 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevent, a clinically significant deficit in the activity, function and response of the patient. Specifically, a therapeutically effective amount will cause one or more of the following: decreased edema; decreased mucous cell metaplasia, decreased mucous production, or a decrease in any other markers as discussed herein or that would be known to one of ordinary skill in the art as it relates to lung disease, airway obstruction, ECH or MCM. The frequency and dosage of the therapy can be titrated by the ordinary physician or veterinarian using standard dose-to-response techniques that are well known in the art.

[0086] As noted above, certain embodiments of the present invention involve the use

BIK polypeptides or mimetic therapy as an efficacious treatment of lung disease or symptoms or treatment of mucous cell metaplasia in the airways or cells for example AECs or other cells in vitro described herein or as known to one of ordinary skill in the art. In particular, a pharmaceutically effective amount of a pharmaceutical composition for treatment of lung disease or symptom is administered to a mammalian patient.

Preferably, of from about 0.1-10 mg/kg per day, and more preferably of from about 1-8 mg/kg per day, and most preferably of from about 2-6 mg/kg per day of the

pharmaceutical composition is administered to a patient.

[0087] The pharmaceutical composition includes an active agent composed of at least one cell delivery agent and at least one cell death agent. In certain embodiments, the cell delivery agent is a cell-penetrating peptide (CPP). A CPP is a peptide vector that can traverse through the plasma membrane barrier without breaching the integrity of the cell, and deliver a desired cargo inside the cell. The range of cargoes that can be delivered intracellularly by CPPs encompasses a broad variety of hydrophilic molecules, such as peptides, proteins, antibodies, imaging agents, DNA and even nanosized entities, including polymer-based systems, solid nanoparticles and liposomes. In certain embodiments, the CPP hereof is preferably a cationic amphipathic peptide leader sequence and, more preferably, a TAT sequence. In certain other embodiments, the CPP is selected from the group consisting of TAT, penetratin, VP22, transportan, synthetic oligoarginines, and combinations thereof. One skilled in the art will appreciate, however, that any vector capable of traversing the plasma membrane barrier without breaching the integrity of the cell and capable of delivering a desired active agent inside the cell may be used in an embodiment of the present invention without departing from the scope thereof. Additionally, a Bik polypedtide or a derivative of a Bik polypeptide can be expressed in cells through a viral vector such as adenovirus, lentivirus, or retrovirus or as part of an acceptable expression vector. We have expressed the whole Bik protein using adenoviral expression vector (figure 3). The same approach would be taken to express parts of Bik (the peptides listed above and other parts of Bik) that show the highest effective killing of mucous cells.

[0088] The pharmaceutical composition for treatment of an immune-mediated disorder hereof also includes a cell death agent capable of stimulating cell death.

[0089] Referring now to figure 7, a proposed IFNy-induced pathway that results in

killing of proliferating NHBECs is illustrated.

100901 The mutation in the BH3 domain of Bik will lack the translocation of Bax or Bak to the ER. Identification of the mutation that fails to translocate Bax or Bak to the ER and/or to interact with phosphorylated ER l/2 or DAPkinase may be useful in the future to identify which protein(s) may be involved in directly interacting with Bik to cause Bax or Bak translocation to the ER. We expect that instillation of the active peptides into allergic mice will cause a reduction of MCM, similar to what we observed in mice instilled with Ad-Bax or Ad-Bik (see Preliminary Results). Once the active peptide is identified, studies to deliver the peptide by aerosolizing it to directly target metaplastic mucous cells will be conducted in the future. Because Noxa was found to enhance Bik induced cell death, we will consider using a combination of Bik and Noxa to possibly have a synergistic reduction of MCM and thereby reduce the amount of peptide used. Noxa enhances Bik-induced cell death. 100911 Cigarette smoke exposure destabilized Bik mRNA rather than affecting promoter activity. Similarly, I Ny-induced Bik expression does not involve the promoter activity. However, in human B cells, TGF-β induces apoptosis by TGF-P-activated Smad transcription factor complexes being recruited to a consensus Smad-binding element in the Bik promoter and directly inducing Bik transcription. Other studies have reported that Bik protein degradation is modified by anti-cancer agents. Bortczomib, a proteasome inhibitor, induces cell death in various cancer cells and primary HA EC's by inhibiting Bik protein degradation. Therefore, Bik expression can be regulated at both the transcriptional and post-transcript ional levels. Since Bik remains suppressed in the lungs of mice even 60 days after recov ery then miRNA or the RNa.se that is activated by CS once induced, remains intact ev en after cessation of CS exposure. Expression of Bik is dependent on a polymorphism variant in the ntronic region of the Bik gene. The common variant encoded by guanine causes reduced expression of Bik while the rare v ariant encoded by adenine causes increased expression. Therefore, this variant can be used to determine the susceptible person who develops mucous cell metaplasia and mucous hypersecretion. In addition, treatment with peptide can be specifically optimized by genotyping indiv iduals who are prone to expressing low levels of Bik due to their genetic polymorphism in the intronic region of Bik .

100921 Modification of Leu within the BH3-domain of Bik to Gly was sufficient to

abolish the interaction between Bik and the 60 k Da DAP fragment. Phosphorylation of Bik at residues 33 (threonine) and 35 (serine) is requi ed for eliciting efficient apoptotic activity. Bik may modify phospho-ER K 1 /2 to interact to DAPK . ERKl/2 or DAPK may phosphorylate Bik to enhance the formation of the B i k-E Rk 1 2- D A PK complex. Bik disrupts the interaction of Bcl-2 and nutrient-deprivation autophagy factor- 1 (NAF-1) to regulate Bik-initiated autophagy, but not Bik-initiated activation of caspases. Therefore, while Bik may promote autophagy by displacing NAF-1 from Bcl-2, Bik promotes an apoptotic cell death by being modified by phospho-ERK 1 /2 to interact with DAPK. 100931 A new paradigm, system, method, compositions and therapies for modulating airways for epithelial remodeling aimed at reducing hyperplastic AECs that are involved in mucous hypersecretion in chronic diseases is disclosed.

Experimental Approach and Methods:

[0094] Identification of the Bik Domain Responsible for Killing NHBECs:

Expression vectors containing different length peptides of the Bik protein were constructed. Constructs will express either the BH3 domain of Bik or will include 5, 10, or 15 amino acids on the N- or C-termini of the BH3 domain. Constructs in which the conserved Leu residue in the BH3 domain is mutated to Ala by site-directed mutagenesis is presented. The constructs for mutant Bik proteins are tested for cell killing along with the construct expressing the wild-type Bik. Constructs that show effective cell killing are then tested by immunofluorescence for their effect on translocating Bax to the ER and their interaction with Bcl-2 or BI-1, depending on results obtained. Peptides are either hydrocarbon stapled to ensure stability, myristylated, or tagged with polyarginine or the TAT protein to ensure entry into cells. The control peptide is randomized to have a sequence different from any of the known BH-3 peptides known to date. Stock solutions at 10-20 mM concentrations are prepared in DMSO. NHBECs are treated with 1, 10, 20, or 100 μιη concentrations of BH3 peptides and analyze the extent of cell death that is induced in NHBECs by this BH3 peptide at 24, 48, and 72 hours. Cell death is assessed by cell counts and by Annexin V staining followed by flow cytometry. The number of cells showing co-localization of Bax with ER are quantified, as denoted by the yellow color as a result of the green calnexin stain overlapping with the red stain for Bax.

[0095] Assessing the Effect of BH3 Peptides in Organ Cultures and in vivo: Because the BH3 domain sequences for Bik in humans and mice are identical and the flanking regions are highly similar, the peptides that are based on the active regions identified for NHBEC killing are used to design peptides that may enhance Bik-induced apoptosis in mouse AECs. The effective peptides are used in the organ culture system with bronchiolar explants to test which of the Bik-derived peptides are effective in resolving IL-9/IL- 13 -induced MCM. Explant cultures from bronchioles and from nasal midseptum are used to successfully identify the most effective antisense oligonucleotides to Bcl-2 mRNA and to introduce Bax by adenoviral infection. Explant cultures are treated with IL- 9 and IL- 13 at 1 ng/ml each for 6 days, and the BH-3 peptides at 1, 10, 20, or 100 um concentrations is added on day 7 of culture as previously described. After an additional 24 and 48 hours of culture, explants are harvested and processed for quantification of MCM as described previously. Furthermore, tissue sections are analyzed for Bax localization to the ER by immunofluorescence as described in Preliminary Studies. To test the therapeutic potential of such an approach, C57B1/6 mice are immunized and exposed to OVA for 5 days as described above to induce maximum MCM. The following day, mice will be instilled with the active peptide that causes cell death in NHBECs at 10 or 100 μΜ in 50 μΐ saline. Mice are instilled with control peptide or with saline only. Two days later, mice are sacrificed, and the extent of MCM is compared between the groups of mice instilled with active Bik peptide, control peptide, or saline only.

[0096] Plasmids, adenoviral constructs, and reagents. Adenoviral expression vectors for

Bik and BikL61G were infected into cells as described previously. Retroviral silencing vector encoding for DAPK shRNA and the control vector were purchased from Origene Technologies, Inc. The suppressing effect of the shRNA was established in HA EC^'s and H 1975 cells, and amplification and purification of pi asm id DNA and packaging of the retroviral particles in Phoeni cells were performed as specified by the manufacturer's instructions. The MAPK extracellular signal-regulated kinase inhibitor 4-diamino-2,3- dicyano-l,4-bis(2-ammophenyltbio) butadiene (U0126) was purchased from EMD

Chemicals Inc. (Darmstadt, Germany).

[0097] Retroviral silencing with DAPK shRNA. Retroviral silencing vectors encoding for DAPK shRNA and the control shRNA (Origene Technologies, Inc. Vectors,

Rockville, MD) were transiently transfected using FuGENE 6 (Roche Bioscience, Palo Alto, CA) into HI 975 to identify the most effective shRNA from 4 constructs. Amplification and purification of pi asm id DNA and packaging of the retroviral particles in Phoenix cells were performed as specified by the manufacturer's instructions. After infection with DAPK or control shRNAs, HI 975 cells were infected with 100 MO I Ad- Bik, and 24 hours later cells were harvested for quantification and Western blot analysis.

[0098] Bronchial brushings. Protocols were approved by the University of New Mexico

School of Medicine and the Lovelace Respiratory Research Institutional Review Boards to obtain all bronchial samples. Bronchial brushings were obtained by bronchoscopy at the University of New Mexico Health Sciences Center. All participants were recruited by advertising in local newspapers and in the University newspaper. Chronic bronchitis was defined as a daily cough with phlegm production for 3 consecutive months, 2 years in a row. Bronchial brushings were obtained from 11 subjects each with chronic bronchitis, and 9 controls with no evidence of lung disease. Demographics of these subjects is described in table 1. Bronchial brushings, performed under local anesthesia with 1% lidocaine, contained 0.4-2 million epithelial cells. At least 60,000 cells from each subject were used for qRT-PCR as described previously.

[0099] Lung autopsy tissues. Autopsy tissues were obtained from the Lung Tissue

Research Consortium (LTRC), NHLBI. The subjects were categorized into four groups based on records obtained by questionnaires administered. For the subgroups classified by chronic bronchitis, the definition of "signs with chronic bronchitis", which is cough and phlegm for 3 consecutive months and for at least 2 years was used. All, who did not answer yes for these questions and answered "do not usually have cough, and do not usually have phlegm" were defined as subjects with no signs of chronic bronchitis.

Subjects with less than 15 pack years of smoking were excluded. The demographic characteristics of the subjects from whom autopsy tissues were used for our studies are shown in table 2.

[00100] Mice and adenoviral infection. Male-specific pathogen-free wild-type C57BL/6 mice were purchased from The Jackson Laboratory. Mice were housed in isolated cages under specific pathogen-free conditions. After a 14-day quarantine period, mice were acclimatized for 8 days and entered into the experimental protocol at 8 - 10 weeks of age. bik+/- mice on C57BL/6 background were provided by Andreas Strasser (Walter and Eliza Hall Institute, Melbourne, Australia). bik+/+ with bik-l- littermates were bred from the respective heterozygote mice at the Lovelace Respiratory Research Institute under specific pathogen- free conditions and genotyped as described previously. All experiments were approved by the Institutional Animal Care and Use Committee and were conducted at Lovelace Respiratory Research Institute, a facility approved by the Association for the Assessment and Accreditation for Laboratory Animal Care International. Mice were exposed to 250 mg/m3 CS or filtered air for 6 hours/day, 5 days/week for 3 weeks or were allowed to recover in air for an additional 8 weeks following 3 weeks of CS exposure. Preparation of lung tissues for histopatho logical examination was performed as described previously. After 3 weeks of exposure to CS mice were anesthetized with isoflurane and intranasally instilled with HA-Ad-Bik or Ad-GFP as a control in a volume of 50ul saline on day 1 and 2 after the last day of exposure. On day 3, six mice from each group were euthanized and right lung tissue harvested and immediately examined for expression of HA protein via Western blotting of protein extracted from lung homogenate. Left lungs were inflated and fixed at 25 mm pressure with zinc formalin for preparing tissue sections and evaluating ECH and MCM.

[00101] Tissue sections were stained with Alcian blue (AB) and periodic acid Schiff or hematoxylin and eosin as described previously. The number of AB positive cells per millimeter of basal lamina was quantified using a light microscope (BH-2; Olympus) equipped with the Image analysis system (National Institutes of Health) as described previously.

[00102] Cells. MAECs were harvested and cultured on Transwell membranes (Corning) after seeding with 4 x 104 or 9 x 104 cells as previously described. Primary HAECs were purchased from Cambrex Bio Science Walkersville, Inc. The immortalized HAECs, AALEB cells, were provided by S. Randell (University of North Carolina Chapel Hill, Chapel Hill, NC) were described previously. The lung cancer cell lines with wild-type or mutant K-ras, NCI-H1299 and NCI-H1975 were purchased from ATCC (HAECs were maintained in bronchial epithelial growth medium supplemented with growth factors as described previously. HI 975 and HI 299 cells were maintained in RPM medium supplemented with 10% serum, 0.01% L-glutamine and 0.01% antibiotics. Cell viability was assessed by trypan blue exclusion.

[00103] SDS-PAGE and immunoblotting. Protein lysates were prepared and analyzed by

Western blotting as described previously. Cytosolic and nuclear fractions were prepared by lysing cells in NP-40 to obtain the cytosolic fraction and extracting the nuclear proteins with a hypertonic extraction buffer (50 mM Hepes, pH 7.8, 50 mM KC1, and 300 mM NaCl) in the presence of protease and phosphatase inhibitors as described previously. The following antibodies were used: goat anti-Bik polyclonal antibody (Santa Cruz

Biotechnology, Inc.), rabbit anti-Bik antibody, rabbit anti-DAPK antibody, rabbit anti- phospho-ER l/2 antibody, and rabbit anti-ER l/2 antibody (Cell Signaling

Technology). Equal protein loading was confirmed by subsequent probing with the mouse monoclonal antibody against actin (Santa Cruz Biotechnology, Inc.).

[00104] Immunofluorescence. Sections of differentiated primary HAECs and of lung tissues were deparaffmized, rehydrated, washed, and, after antigen retrieval. Sections were incubated with a 1 : 1000 dilution of anti MUC5 AC antibody (MAB2011 , Chemicon) at 4 °C overnight followed by 1 :500 dilution of secondary donkey anti-mouse-biotin antibody (Jackson Immunoreaserch) at room temperature for 1 hour. Vectastain® ABC (Vector Laboratories, Inc., Ca) and streptavidin 649 dye (Jackson Immunoresearch) was used for detection and mounting in DAPI FluormountTM-G (SouthernBiotech).

Immunofluorescence was imaged using Axioplan 2 (Carl Zeiss, Inc.) with a Plan-Neofluor 40x/0.75 air objective and a charge-coupled device camera (Hamamatsu Photonics, Japan) with the acquisition software Slidebook 5.0 (Intelligent Imaging Innovation).

[00105] Pull-down assays. Pierce® Crosslink Immunoprecipitation Kit (Thermo

Scientific, Rockford, IL) was used to cross-link 10 μg of purified anti-Bik or anti-p- ERKl/2 antibody to protein A/G beads using disuccinmidyl suberate as described by the manufacturer. Bik-associated proteins were immunoprecipitated by incubating protein lysates prepared from Ad-Bik-, or Ad-BikL61G- infected AALEB or HI 975 cells with gentle mixing at 4°C overnight. After repeated washes, proteins bound to the Bik or p- EPv l/2 antibody on beads were eluted with 0.2 ml of ImmunoPure Elution buffer (Thermo Scientific) and analyzed by Western blotting using anti-phospho-ER l/2, anti- Bik, anti-HA and anti-DAPK antibodies.

[00106] qRT-PCR. RNA isolated using the RNeasy Micro kit (Qiagen) eluted from the columns subjected to quantitative real-time PCR on the ABI PRISM 7900HT Real-Time PCR System using the One-Step RT-PCR Master Mix (Applied Biosystems). The primer/probe sets (Applied Biosystems) were distributed into each well in duplicates, and target mRNAs were amplified by PCR in 20- μΐ reactions. Preamplification efficiency was assessed by performing amplification of non-amplified cDNA with TaqMan Gene Expression Assays (Applied Biosystems). For all reactions, CT values >37 were eliminated for evaluation of preamplification efficiency. Uniform preamplification was demonstrated by a AACT value of -1.5 to 1.5 when comparing the CT values of each gene amplified from preamplified and nonamplified cDNA as described previously. Because all results were derived from the linear amplification curve, the use of the AACT method ensures that only mRNA amplification within the linear range was compared.

Normalizing mRNA levels using 18S rRNA or CDKN1B showed similar results.

[00107] Bik promoter activity and Bik mRNA stability. Human Bik promoter was

amplified by PCR from normal colon genomic DNA and inserted into the pGL3 -Basic Firefly luciferase reporter plasmid (Promega, Madison, WI). MCF7 cells were transfected with BIK promoter reporter plasmids and a control plasmid expressing the Renilla luciferase (Promega) and incubated in the presence or absence of 100 nmol/L fulvestrant for 48 hours, and expression of reporter genes was determined by the Dual Luciferase assay (Promega). Stability of the Bik mRNA transcripts was examined by determining the time-dependent decrease in Bik mRNA amount after terminating cellular mRNA synthesis with 5,6-dichloro-l-B-D-ribofuranosylbenzimidazole (DRB) by qRT-PCR as described previously. [00108] Statistical analysis. Grouped results were expressed as means ± SEM. Data were analyzed using statistical analysis software (Statistical Analysis Software Institute). Results grouped by time point and genotype were analyzed using two-way analysis of variance. When significant main effects were detected (P < 0.05), Fisher's least significant difference test was used to determine the differences between groups. A P- value of 0.05 was considered to indicate statistical significance.

[00109] Bik, a protein localized to the endoplasmic reticulum (ER), is the major regulator of the IFNy-induced cell death pathway. Noxa enhances Bik-induced apoptosis in proliferating AECs. Modulating Bik is useful to control allergen induced MCM and help reduce symptoms associated with excessive mucous secretion in chronic pulmonary/lung diseases. Bik expression is a biomarker for the health of an individual and biomarker for treatment of lung disease and MCM.

Table 1

Demographics of bronchial brushing donors

Controls n = 9 Chronic Bronchitics n

AgeA 31.4 ± 12.6 36 ± 9.23

Gender, M/F 4/5 5/6

Smoking Status, 0/9 2/9

Y/N

FEV1 %pred.A 96.0 ± 7.7 102.09 ± 9.4

FVC %pred.A 99.7 ± 9.9 107.4 ± 10.2

FEV1/FVCA 81.1 ± 2.4 79.6 ± 6.0

M = male; F = female. AMean ± SD.

Table 2

Demographics of lung tissue donors

Never Smokers n Current Smokers Former Smokers Former Smokers

= 5 n = 4 n = 6 n = 6

Chronic No Yes No Yes

Bronchitis

AgeA 61 .6 ± 10.9 61 ± 4.2 64 ± 10 60 ± 8.9

Gender, M/F 2/3 3/1 4/2 6/0

Smoking in 0 41 .8 ± 27.4 38.8 ± 16.5 49.7 ± 24.5

PYA

Stop N/A No 1 1.2 ± 6.8 8.7 ± 8.4

SmokingA

FEV1 %pred.A 67 ± 30.1 64.5 ± 18.6 51 ± 36.9 35.2 ± 18.9

FVC %pred.A 81 .4 ± 21.9 71.3 ± 16.7 73.2 ± 25.8 65.3 ± 16.4

FEV1/FVCA 0.8 ± 0.28 0.89 ± 0.13 0.64 ± 0.28 0.48 ± 0.20

M = male; F = female. AMean ± SD. [00110] REFERENCES

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[00111] Embodiments of the present invention have been described in terms of preferred embodiments, however, it will be appreciated that various modifications and improvements may be made to the described embodiments without departing from the scope of the invention. The entire disclosures of all references, applications, patents, and publications cited above and/or in the attachments, and of the corresponding application(s), are hereby incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1 A method of inducing anti-cell proliferation activity, and/or pro-apoptotic activity in a subject, comprising administering to the subject a therapeutic effective amount of a Bik polypeptide or a Bik polypeptide derivative having an altered amino acid sequence, said polypeptide comprising: a) SEQ ID NO: 1-18; or polypeptides having sequence that is between 91% and 99% identical to SEQ ID NO: 1-18 or any combination thereof.

2. The method of claim 1, wherein the subject is an animal.

3. The method of claim 2 wherein the subject is a human.

4. The method of claim 2, wherein the human has a proliferative cell disorder.

5. The method of claim 3, wherein the proliferative cell disorder is mucous cell metaplasia or epithelial cell hyperplasia.

6. The method of claim 1, wherein the polypeptide is comprised in pharmacologically acceptable excipient or diluent.

7. The method of claim 1, wherein the polypeptide is complexed with a lipid.

8. The method of claim 1, further defined as a method of preventing growth of a cell in an individual.

9. An isolated antibody that specifically binds to the polypeptide of SEQ ID NO: 1-18 or a derivative thereof.

10. The antibody of claim 9 which is a monoclonal antibody.

11. The antibody of claim 9 which is a humanized antibody.

12. An antigen-binding fragment of the antibody of claim 9.

13. The antibody of claim 9 which is labeled.

14. A method of treating an allergen induced disease of the respiratory system in a patient comprising the steps of administering a pharmaceutical composition of claim 16 to the patient, said composition including a cell delivery agent and a cell death agent, in an amount sufficient to provide a clinically observable improvement in the disease symptoms of the patient.

15. The method of claim 14 wherein said composition is dispersed in a pharmaceutically acceptable carrier.

16. A composition comprising a polypeptide of SEQ ID NOs 1-18 or a derivative thereof.

17. The composition of claim 16 further comprising a cell delivery agent.

18. The composition of claim 17 wherein said cell delivery agent is a cationic amphipathic peptide leader sequence.

19. A pharmaceutical composition for treating an immune-mediated disease comprising: a pharmaceutically effective amount of at least one cell delivery agent; a pharmaceutically effective amount of a composition of claim 16; and a pharmaceutically acceptable carrier.

20. The composition of claim 16 formulated as an aerosol.

21. The method of claim 14 wherein the pharmaceutical composition is provided into the lung of the patient by inhalation.

22. The method of claim 14 wherein the pharmaceutical composition is given to patients with specific susceptibility variants that make them express reduced levels of Bik.