RAPID COMMUNICATION
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Abstract |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Airway inflammation, hyperreactivity, increased number of goblet cells, and mucus overproduction characterize asthma. Respiratory challenge with ovalbumin (OVA) of sensitized mice has been shown by several laboratories to cause pulmonary pathology similar to that observed in human allergic asthma. Recently, interleukin (IL)-13 has been shown to be a central mediator in this process. Because the airways of healthy mice have few, if any, mucus-producing cells, an increase in the number of these cells likely reflects induction of mucin-gene expression. The purpose of this study was to identify mucin genes induced as a result of airway goblet-cell metaplasia (GCM) in mice sensitized and challenged with OVA or in mice treated with IL-13 alone. BALB/c mice were sensitized by intraperitoneal injection (Days 0, 4, 7, 11, and 14) and intranasal instillation (Day 14) of 100 µg of OVA in saline, and then challenged by intranasal instillation (Days 25, 26, and 27) of the same. IL-13-treated mice received 5 µg of IL-13 by intranasal instillation on three consecutive days. Control mice were given saline alone. All mice were studied 24 h after the last challenge. Histologic analysis of the lungs revealed both a striking peribronchial and perivascular lymphocytic and eosinophilic inflammation and airway GCM in OVA-treated mice, and also airway GCM without inflammation in IL-13-treated mice. Northern blot analysis of lung RNA demonstrated (1) expression of Muc-5/5ac messenger RNA (mRNA) in OVA-treated and IL-13-treated mice, but not in control mice; (2) expression of Muc-1 mRNA at comparable levels in all mice regardless of treatment; and (3) no expression of Muc-2 or Muc-3 mRNA in control or treated mice. Western blot analysis demonstrated the expression of Muc-5/5ac protein (both apomucin and glycosylated mucin) in lung lysates of OVA-treated (but not control) mice, and also the expression of Muc-5/5ac mucins in the bronchoalveolar lavage fluid of OVA-treated and IL-13-treated mice. These findings demonstrate that airway GCM is associated with the induction of pulmonary expression of Muc-5/5ac mRNA and mucin in murine models of allergic asthma.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The respiratory tract epithelium of mammals is protected
by mucus, a viscoelastic gel normally produced at relatively
low levels. Overproduction of mucus occurs in chronic airway diseases, causing airway obstruction and contributing
to the morbidity and mortality associated with asthma,
bronchitis, and cystic fibrosis (1, 2). The major macromolecular components of mucus are mucin glycoproteins
(mucins), and these large, highly glycosylated macromolecules have protein backbones encoded by MUC genes (reviewed in References 3 and 4). Of the nine currently identified human MUC genes, seven
MUC1, MUC2, MUC4,
MUC51, MUC5B, MUC7, and MUC8are normally expressed in the human upper and/or lower respiratory tract
(5, 6; and reviewed in Reference 7).
Airway inflammation, hyperreactivity, and increased numbers of goblet cells with mucus overproduction (2, 8- 11) characterize asthma, the most common chronic disease in children (12). Recently, several laboratories have shown that respiratory challenge with ovalbumin (OVA) in mice previously sensitized to OVA causes physiologic and pathologic changes similar to those seen in human allergic asthma, including airway hyperreactivity, airway inflammation, airway goblet-cell metaplasia (GCM)2 with mucus production, and increased immunoglobulin E (IgE) production (13). T-helper 2 (Th2) cytokines are essential in the pathogenesis of allergen-induced asthma in murine models (reviewed in References 18 and 19), and interleukin (IL)-13 has now emerged as a central mediator (20, 21). However, our understanding of the regulation of airway mucus production in health and disease is limited. It is generally accepted that an increase in mucus production will require increased expression of mucin genes (22). However, an association of mucin gene expression with specific airway disease has not been determined. The purpose of this study was to identify mucin genes associated with GCM both in mice sensitized and challenged with OVA and in mice whose airways were directly exposed to IL-13 alone.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Mice
Male BALB/c mice, 4 to 6 wk old, were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and housed in an environmentally controlled pathogen-free animal facility accredited by the American Association for the Accreditation of Laboratory Animal Care. The handling of mice was carried out in accordance with institutional protocols, based on National Institutes of Health guidelines for laboratory animal research. Mice were observed for a week prior to experimental manipulation. To facilitate pulmonary aspiration with intranasal instillation, mice were lightly anesthetized by methoxyflurane (Mallinckrodt Veterinary, Inc., Mundelein, IL) inhalation.
OVA Sensitization and Challenge
The sensitization and challenge protocol used, an adaptation of two recently reported procedures (15, 16), is briefly described below and in Figure 1A. Twenty-four mice were evenly divided into two groups: (1) OVA-treated mice were sensitized and challenged with OVA in saline, and (2) control mice were given saline only. Mice were sensitized by intraperitoneal injection (Days 0, 4, 7, 11, and 14) and intranasal instillation (Day 14), and challenged by intranasal instillation (Days 25, 26, and 27). Intraperitoneal doses consisted of 100 µg of crystalline OVA (Grade V, No A-5503; Sigma Chemical Corporation, St. Louis, MO) in 0.1 ml of saline (0.9% sodium chloride injection, USP; Abbot Laboratories, North Chicago, IL). Intranasal doses contained 100 µg of OVA in 0.05 ml of saline. Control mice received equivalent volumes of saline only. Mice from both groups were killed by CO2 asphyxiation, 24 h after the last intranasal instillation (Day 28).
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IL-13 Treatment
Ten mice were divided evenly into two groups: IL-13-treated and control mice. IL-13-treated mice were given 5 µg of recombinant murine IL-13 (R&D Systems, Inc., Minneapolis, MN) in 0.05 ml of saline by intranasal instillation on each of three consecutive days (Figure 1B). Control mice were given equivalent volumes of saline only. Mice from both groups were killed by CO2 asphyxiation, 24 h after the last intranasal instillation.
Bronchoalveolar Lavage and Histologic Analysis of Lungs
Bronchoalveolar lavage (BAL) and histologic analysis of
lungs were performed on one half of the mice from each
study group. Immediately after death, the chest was opened
and the trachea cannulated with a small catheter. BAL
was performed by instillation of 1 ml of normal saline. The
recovered fluid was kept on ice and centrifuged at 900 × g
to separate cells from supernatant. The supernatant was
stored at 
70°C and subsequently used for Western blot
analysis. Lungs were then removed from the chest, inflated with 3 ml of 10% phosphate-buffered formalin using
the intratracheal cannula, and fixed by immersion in 10%
formalin. Tissues were subsequently embedded in paraffin, sectioned, and stained with hematoxylin and eosin to
evaluate general morphology, Giemsa to demonstrate eosinophils, or Alcian blue/periodic acid-Schiff (AB/PAS) to
determine the presence of mucin glycoconjugates (23).
Northern Blot Analysis
Lungs, heart, stomach, and small intestine from the remaining mice were frozen immediately in liquid nitrogen
and stored at 70°C. For RNA extraction, tissues were
homogenized in TRIzol (Life Technologies, Inc., Gaithersburg, MD), and total RNA was isolated according to
the manufacturer's instructions. Aliquots of RNA (10 µg)
from each organ were subjected to electrophoresis in an
1% agarose gel containing 0.4 M formaldehyde. RNA
was transferred by capillary blotting to GeneScreen Plus
nylon membranes (NEN Life Science Products, Inc., Boston, MA) and cross-linked with ultraviolet light using a
Stratalinker (Stratagene, La Jolla, CA). Prehybridization
and hybridization of the blots and preparation of [32P]-
labeled complementary DNA (cDNA) probes were performed as previously described (24). After an overnight
hybridization, membranes were washed twice with 2× saline sodium citrate (SSC)/0.1% sodium dodecyl sulfate
(SDS) at room temperature for 30 min and once with 0.1×
SSC/0.1% SDS at 60°C for 10 min. For autoradiography, membranes were exposed to Kodak X-ray films in the
presence of intensifying screens at 70°C for 24 to 48 h.
cDNA Probes
cDNA probes were generously provided by colleagues. Murine Muc-1 (pMuc2TR) (25) was provided by Dr. Sandra Gendler (Mayo Clinic, Scottsdale, Arizona); murine Muc-2 (D2) (26) and rat Muc-3 (RMUC 176) (27) by Dr. James Gum (University of California at San Francisco); and murine Muc-5 (pMGM1) (28) by Dr. Samuel Ho (University of Minnesota).
Immunochemical and Lectin Analyses
BAL fluid and tissues solubilized in lysis buffer (120 mM NaC1/1% deoxycholate [wt/vol]/1% Triton X-100 [vol/vol]/ 0.1% SDS [wt/vol]/50 mM Tris HCl, pH 8.0) were analyzed for protein by the Bradford microassay (BioRad, Hercules, CA). Aliquots were subjected to electrophoresis on 1% agarose gels, transferred to polyvinylidene difluoride (PVDF) membranes, and analyzed for Muc-5ac protein and mucin and for glycoproteins as previously described (24). For Western blot analysis, membranes were immunostained with rabbit monospecific anti-MUC5:TR-3A antibodies and horseradish peroxidase-conjugated goat antirabbit antibody. For lectin analysis, membranes were stained with wheat germ agglutinin (WGA) using the Vectastain horseradish peroxidase kit (Vector Laboratories, Burlingame, CA) with 3,3',5,5'-tetramethylbenzidine as substrate.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Analysis of OVA-Treated Mice
Histologic analyses. Several significant differences were evident upon comparison of lung sections from OVA-treated and control mice. First, striking peribronchial and perivascular lymphocytic and eosinophilic inflammation was observed in OVA-treated mice (Figure 2A) but not in control mice (Figure 2B). Second, markedly thickened airway epithelium (as measured by the height above the nuclei) was observed in OVA-treated mice (Figure 2A) in contrast to the typically thin airway epithelium of control mice (Figure 2B). This was particularly evident in the larger airways. And third, goblet cells that stained with AB/PAS were abundant in the airways of OVA-treated mice (Figures 2C and 2E) and absent in the airways of control mice (Figures 2D and 2F).
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Evaluation of mucin gene expression. In order to evaluate the expression of mucin genes in the lungs of OVA-treated and control mice, mucin mRNA levels were examined by Northern blot analysis using murine Muc-1, Muc-2, Muc-5, and rat Muc-3 cDNA probes. RNA samples isolated from murine heart, stomach, and small intestine were used as control tissues for probe specificity. Muc-1 mRNA (Figure 3B) was detected at comparable levels in the lungs of OVA-treated (lanes 1-4 ) and control (lanes 5-8) mice. Muc-1 mRNA was also detected in the stomach (lane 10) but not the heart (lane 9) or small intestine (lane 11) of control mice, as expected (25). Muc-2 mRNA (Figure 3C) was not detected in the lungs of either OVA-treated (lanes 1-4 ) or control (lanes 5-8) mice. Muc-2 probe hybridized to several bands in RNA from small intestine (lane 11 ) of control mice. Similar polymorphism has been reported for Muc-2 mRNA from rat intestines (29). Likewise, the Muc-3 probe hybridized to small intestine mRNA as previously reported (27) but not to lung mRNA of either control or OVA-treated mice (data not shown). On the contrary, marked expression of Muc-5 mRNA (Figure 3D) was observed in the lungs of OVA-treated (lanes 1-4 ) but not control (lanes 5-8) mice. Muc-5 mRNA was well-expressed as a polydisperse pattern in the stomach (lane 10) as previously reported (28), but not in the heart or small intestine (lanes 9 and 11) of control mice.
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Analysis of IL-13-Treated Mice
Histologic analyses. A comparison of the lung sections from IL-13-treated and control mice showed several significant differences. Markedly thick airway epithelium was observed in IL-13-treated mice (Figure 4A), whereas control mice exhibited a typically thin airway epithelium (Figure 4B). Goblet cells that stained with AB/PAS were abundant in the airways of IL-13-treated mice (Figures 4C and 4E) and absent in the airways of control mice (Figures 4D and 4F). GCM was more prominent in the larger airways (data not shown). Importantly, the striking peribronchial and perivascular lymphocytic and eosinophilic inflammation observed in the lungs of OVA-treated mice (Figure 2A) was not observed in the lungs of IL-13- treated mice (Figure 4A).
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Evaluation of mucin gene expression. The expression of mucin mRNA in the lungs of IL-13-treated and control mice was examined by Northern blot analysis. Muc-1 mRNA (Figure 5B) was detected at comparable levels in the lungs of IL-13-treated (lanes 1-3) and control (lanes 4-6 ) mice. Neither Muc-2 nor Muc-3 mRNA was detected in the lungs of control or IL-13-treated mice (data not shown). Marked expression of Muc-5 mRNA (Figure 5C) was detected in the lungs of IL-13-treated (lanes 1-3) but not control (lanes 4-6 ) mice.
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Expression of Muc-5 Protein and Mucin in OVA-Treated and IL-13-Treated Mice
An increase in mRNA expression typically, but not always (30), translates into increased production of a gene product. To determine whether the Muc-5 mRNA expression observed in the airways of OVA-treated and IL-13-treated mice was reflected in the increased expression of Muc-5 protein and glycoprotein, we carried out Western blot analysis of murine tissues using affinity-purified polyclonal anti-MUC5 antibodies. These antibodies recognize both apo (unglycosylated) and mature (glycosylated) human MUC5 mucins (24) and were raised against a twenty-two amino acid sequence of the TR-3A domain of MUC5 (31, 32), which is 82% identical to the murine Muc-5 TR-3a sequence (28). This high degree of conservation suggested that the anti-MUC5:TR-3A antibodies might recognize murine Muc-5/5ac mucins. Western blot analyses of murine gastric tissue homogenates, which express the Muc-5 protein (28), demonstrated that the affinity-purified polyclonal anti-MUC5:TR-3A antibodies recognized murine Muc-5 protein (data not shown). Lung lysates from control mice showed no immunostaining (Figure 6A, lane 1 ), whereas two high molecular-weight (MW) bands immunostained in the lung homogenates of OVA-treated mice (Figure 6A, lane 2). We established the identities of the immunostained bands as apomucin or glycosylated mucin using WGA lectin staining (Figure 6B), as WGA recognizes N-acetylglucosamine and sialic acid residues in oligosaccharide chains (33). Band I, the immunoreactive lower MW band observed in homogenates of the stomach (data not shown) and of lungs of OVA-treated mice (Figure 6A, lane 2), was identified as Muc-5 apomucin because it did not react with WGA lectin (Figure 6B, lane 2). Band II in the lung homogenates of OVA-treated mice (Figure 6A, lane 2) was identified as Muc-5 mucin because it both immunostained with anti-MUC5 antibody and stained with WGA lectin. Neither bands I nor II was detected by immunostaining when the antibody was pre-incubated with TR-3A peptide (data not shown). Band III, which reacted strongly with WGA (Figure 6B, lane 2) is an unidentified high MW glycoconjugate present in the lung lysates of OVA-treated, but not control, mice. Secreted Muc-5 mucin glycoprotein was present in the BAL fluid of OVA-treated (Figure 7, lanes 3 and 4 ) and IL-13- treated (lane 1 ) mice but not in BAL fluid from control mice (lanes 2, 5, and 6 ).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Airway inflammation, hyperreactivity, and increased numbers of goblet cells concomitant with increased mucus production (8) characterize asthma, the most common chronic disease in children (12). The determinants of the latter phenotype are not yet well understood, although increased numbers of goblet cells is the most characteristic finding even in newly diagnosed asthmatics (34), and overproduction of mucus leads to airway obstruction and contributes to the morbidity and mortality in asthma (2, 35).
Recently developed mouse models of allergic asthma have shown that mice sensitized and challenged with allergens develop characteristics similar to those observed in human allergic asthma. These include pulmonary lymphocytic and eosinophilic inflammation, airway hyperreactivity (13, 14, 36), an increased number of goblet cells, and mucus obstruction (15, 16, 36, 37). There are two significant differences between murine and human airways (38): Mice normally exhibit few or no mucus-producing cells in their lower airway epithelium, and mice lack submucosal glands. Nevertheless, in the absence of submucosal glands, mice do provide useful in vivo model systems for dissecting the role of GCM and mucin gene regulation in the airway epithelium. Although submucosal glands have long been implicated in mucus obstruction in asthma (39), a recent reassessment has led to a re-emphasis of the role of goblet cells in mucus obstruction (22). Further, the absence of mucus-producing cells in normal murine lower airway epithelium and their appearance in OVA-treated (Figure 2) and IL-13-treated (Figure 4) murine airways suggest a metaplastic change in the murine airway epithelium, reflecting differentiation of epithelial cells that lead to GCM and subsequent mucus overproduction.
In this study we have established a reproducible model of OVA-induced asthma in BALB/c mice using a protocol that combined salient features of procedures evaluated by Blyth and collegues (15) and Zhang and associates (16). Specifically, we decreased the number of sensitization steps, did not include an adjuvant during sensitization, and performed respiratory challenge by intranasal instillation of the allergen under light anesthesia. Pulmonary aspiration of OVA was sufficient to cause marked histologic changes (peribronchial and perivascular inflammation, epithelial thickening, and airway GCM) comparable to those reported earlier with intratracheal challenge (15). Pulmonary aspiration of IL-13 alone also caused airway epithelial thickening and GCM, as reported earlier with intratracheal challenge (20). The absence of airway inflammation after short-term exposure to IL-13 is interesting, as transgenic mice that chronically overexpress IL-13 in their airways clearly exhibit lymphocytic and eosinophilic inflammation concomitant with GCM (40).
Increasing evidence supports the concept that GCM and mucus production are directly affected by cytokines that regulate immune functions in mice. GCM and mucus production appear to be independent of B cells and immunoglobulins (41) and dependent on Th2 cytokines. IL-4 was implicated initially, as blockage of the IL-4 receptor in mice sensitized and challenged with antigen markedly decreased airway GCM (18, 19). Subsequently, IL-4 was shown to play an indirect role in mucus production through the recruitment of Th2 cells to the lungs and induction of inflammation (42). Recently, the Th2 cytokine, IL-13, was demonstrated to be a central mediator in the GCM/mucus obstruction phenotype evidenced in the mouse model of allergic asthma (20, 21).
In this study we have shown that airway GCM correlates with induction of Muc-5 mRNA and Muc-5 protein (both apomucin and mucin glycoprotein) expression in a mouse model of allergic asthma generated by OVA sensitization and challenge. We have also demonstrated a similar correlation between airway GCM and Muc-5 gene and protein expression by directly exposing murine airways to IL-13. Further, we have shown that neither Muc-2 nor Muc-3 mucin genes is induced in these models and that Muc-1 expression is unchanged. Rankin and coworkers have shown that IL-4 transgenic mice, in which overexpression of IL-4 is localized to the airways (43), exhibit airway GCM and induction of Muc-5, but not Muc-2, mRNA expression (44). These IL-4 effects may be the result from the recruitment of Th2 cells, which release IL-13. Nadel and coworkers have recently reported that direct exposure of murine airways to IL-4 increases Muc-5 gene expression and AB/PAS staining (45), although the specific Muc mucins responsible for increased AB/PAS staining were not identified. Together, available data indicate that upregulation of the Muc-5 gene plays a role in airway GCM and in the overproduction of lung mucus in mouse models of asthma. These models may prove relevant to human asthma, as MUC5 mRNA (6, 46) and MUC5 protein (47) are localized to goblet cells in the lower respiratory tract. In addition, MUC5 mucin glycoprotein has been biochemically isolated from lung mucus from a patient with bronchial asthma (31, 32) and immunochemically identified in lung mucus from asthmatic patients and in pooled secretions from healthy individuals (48, 49).
It is becoming increasingly evident that differential regulation of mucin genes occurs in respiratory tract tissue (57). Muc-5 mRNA expression is markedly increased in the lungs of OVA-treated (Figure 3D) and of IL-13-treated (Figure 5C) mice. By contrast, Muc-2 mRNA expression is not detected in the lungs of OVA-treated (Figure 3C) or IL-13-treated mice (data not shown), IL-4 transgenic mice (44), or IL-4-treated mice (45), although it is induced in rat lungs after exposure to SO2 and the Sendai virus (50). Conversely, both MUC2 and MUC5 mRNA expression increase dramatically in human airway tissue explants exposed to lipopolysaccharides (51). The lack of induction of Muc-3 mRNA in OVA- and IL-13-treated mice probably reflects its lack of pulmonary expression; MUC3 appears to be absent in normal human airways (6). That Muc-1 mRNA is expressed at similar levels in control and OVA-treated mice is not unexpected, as alterations in MUC1 expression have been reported primarily in breast (52) and pancreatic (53) carcinomas. Whether the murine homologues of other MUC genes are also upregulated in OVA-treated mice will be determined as additional murine mucin genes are identified. Indeed, an unidentified murine glycoconjugate was identified by WGA-lectin staining in the lung lysates of OVA-treated mice (Figure 6B, lane 2). This may be Muc-4 mucin (expressed and secreted in normal rat airway epithelium [54]) or an unidentified murine mucin.
The mechanisms that govern mucin gene regulation and GCM are most likely complex (57). Although IL-13 has been shown to be a central mediator, other mediators (such as IL-5 and leukotrienes) may also play a role in generating GCM and mucus production in murine airways. Transgenic mice that overexpress the Th2 cytokine, IL-5, in their lungs exhibit increased mucus in their airway epithelium (55). Further, GCM and mucus production are blocked in the presence of specific inhibitors of 5-lipoxygenase and 5-lipoxygenase-activating protein (56). Nevertheless, the identification of Muc-5 as a major gene and gene product in GCM should further encourage studies directed toward understanding the induction and reversal of GCM at a molecular level. Such studies will elucidate mechanisms underlying human airway remodeling and prove relevant to the successful treatment of asthma.
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Footnotes |
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Address correspondence to: Mary C. Rose, Ph.D., or Franco M. Piazza, M.D., Children's Research Institute, Children's National Medical Center, 111 Michigan Ave. NW, Washington, DC 20010. E-mail: MRose{at}cnmc.org or FPiazza{at}cnmc.org
(Received in original form April 14, 1999 and in revised form December 1, 1999).
Abbreviations: Alcian blue/periodic acid-Schiff stain, AB-PAS; bronchoalveolar lavage, BAL; complementary DNA, cDNA; goblet-cell metaplasia, GCM; immunoglobulin, Ig; interleukin, IL; messenger RNA, mRNA; molecular weight, MW; ovalbumin, OVA; polyvinylidene fluoride, PVDF; sodium dodecyl sulfate, SDS; saline sodium citrate, SSC; T helper 2, Th2; wheat-germ agglutinin, WGA.1 Human and murine mucin genes are designated MUC and Muc-, respectively. The MUC5/5AC gene is referred to in the literature both as MUC5 and MUC5AC. For simplicity, we have used MUC5 and Muc-5 in the text.
2 The term GCM is used throughout the manuscript. Murine airways lack goblet cells; thus, the appearance of goblet cells reflects metaplasia, not hyperplasia, as discussed in Reference 15.
Acknowledgments: This work was supported by The Board of Lady Visitors at CNMC, a Children's Research Institute RAC grant to F.M.P., and in part by NIH RO1 grant HL33152 to M.C.R. M.Z.A. gratefully acknowledges the support of Dr. Robert Fink throughout his fellowship training in Pediatric Pulmonology. The authors also thank Dr. Sandra Gendler, Dr. James Gum, and Dr. Samuel Ho for kindly providing rodent mucin probes, and Dr. Nancy Noben-Trauth and Dr. Bhanu Rajput for critical evaluation of this manuscript.
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References |
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Abstract
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Materials and Methods
Results
Discussion
References
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