Evaluating the effects of a neonatal hypoglycemia bundle on NICU admission and exclusive breastfeeding.

OBJECTIVE: Early feeding, skin-to-skin contact, and dextrose gel have been independently shown to promote breastfeeding and decrease NICU admission for neonatal hypoglycemia. We combined these interventions to decrease NICU admissions for asymptomatic hypoglycemia and increase exclusive breastfeeding rates. PROJECT DESIGN: The IHI Model for Improvement was used to design a bundle including feeding within 1 h of birth, 1 h of uninterrupted skin-to-skin within 2 h of birth, and administration of buccal 40% dextrose gel for hypoglycemic infants. RESULTS: Utilization of dextrose gel was 94% following implementation. There were no trends in exclusive breastfeeding at discharge or NICU admissions for asymptomatic hypoglycemia. Post hoc multivariate analysis identified cesarean delivery as an independent risk factor for compliance failure and failure of exclusive breastfeeding but not for NICU admission. CONCLUSIONS: Despite high compliance with dextrose gel utilization, there was no change in exclusive breastfeeding at discharge or NICU admission rates for asymptomatic hypoglycemia.

Lung mucin production is stimulated by the air pollutant residual oil fly ash.

Human and animal exposure to particulate air pollution is correlated with airway mucus hypersecretion and increased susceptibility to infection. Seeking clues to the mechanisms underlying this pathology, we examined the effect of the particulate air pollutant residual oil fly ash (ROFA) on production of the major component of mucus, mucin, and the major antibacterial protein of the respiratory tract, lysozyme. We found that following in vitro exposure to ROFA, epithelial cells showed an increase in mucin (MUC5AC) and lysozyme (LYS) steady state mRNA. This upregulation was controlled at least partly at the level of transcription as shown by reporter assays. Experiments testing the ability of the major components of ROFA to mimic these effects showed that vanadium, a metal making up 18.8% by weight, accounted for the bulk of the response. A screen of signaling inhibitors showed that MUC5AC and LYS induction by ROFA are mediated by dissimilar signaling pathways, both of which are, however, phosphotyrosine dependent. Recognizing that the ROFA constituent vanadium is a potent tyrosine phosphatase inhibitor and that mucin induction by pathogens is phophotyrosine dependent, we suggest that vanadium-containing air pollutants trigger disease-like conditions by unmasking phosphorylation-dependent pathogen resistance pathways.

Allergen-induced IL-9 directly stimulates mucin transcription in respiratory epithelial cells.

A hallmark of asthma is mucin overproduction, a condition that contributes to airway obstruction. The events responsible for mucin overproduction are not known but are thought to be associated with mediators of chronic inflammation. Others have shown that T-helper 2 (Th2) lymphocytes are required for mucous cell metaplasia, which then leads to mucin overproduction in animal models of allergy. We hypothesized that Th2 cell mediators are present in asthmatic airway fluid and directly stimulate mucin synthesis in airway epithelial cells. Results in cultured airway epithelial cells showed that samples of asthmatic fluid stimulated mucin (MUC5AC) synthesis severalfold more potently than non-asthmatic fluid. Consistent with this, lavage fluid from the airways of allergen-challenged dogs stimulated mucin synthesis severalfold more potently than that from non-allergen-challenged dogs. Fractionation of dog samples revealed 2 active fractions at <10 kDa and 30-100 kDa. Th2 cytokines in these molecular weight ranges are IL-9 (36 kDa), IL-5 (56 kDa), and IL-13 (10 kDa). Antibody blockade of ligand-receptor interaction for IL-9 (but not IL-5 or IL-13) inhibited mucin stimulation by dog airway fluid. Furthermore, recombinant IL-9, but not IL-5 or IL-13, stimulated mucin synthesis. These results indicate that IL-9 may account for as much as 50-60% of the mucin-stimulating activity of lung fluids in allergic airway disease.

Long-lasting effects of chronic ozone exposure on rat nasal epithelium.

Ozone, the principal oxidant pollutant in photochemical smog, causes airway epithelial injury in the upper and lower respiratory tract of laboratory animals. We have recently reported that long-term inhalation exposure to ozone causes mucous-cell metaplasia (MCM) in the surface epithelium lining the nasal airways of F344 rats. The principal objective of the present study was to determine the persistence of ozone-induced MCM in the nasal epithelium after the end of a chronic exposure. Male F344/N rats were exposed to 0, 0.25, or 0.5 ppm ozone, for 8 h/d, 7 d/wk for 13 wk. Animals were killed 8 h, 4 wk, or 13 wk after the end of the chronic exposure. Ozone-related alterations in the nasal epithelium were qualitatively and quantitatively characterized through histochemistry, image analysis, and morphometric techniques. Some rats were exposed for an additional 8 h to 0.5 ppm ozone at 13 wk after the end of the chronic exposure to determine whether previous ozone exposure results in persistent changes in the sensitivity of nasal epithelium to acute injury. At the end of the chronic exposure, hyperplasia was present in the nasal epithelium of rats exposed to 0.25 and 0.5 ppm ozone. By 13 wk postexposure, this proliferative alteration was still evident only in the rats exposed to 0.5 ppm ozone. Ozone-induced MCM with associated intraepithelial mucosubstances was evident only in the nasal tissues of rats exposed to 0.5 ppm ozone. Though attenuated, these alterations in the nasal mucous apparatus were still detectable at 13 wk after the end of the exposure. At this same time after the chronic exposure, an acute (8 h) exposure to 0.5 ppm ozone induced an additional increase of mucosubstances in the nasal epithelium of rats previously exposed to 0.5 ppm ozone, but not in rats chronically exposed to 0 or 2.5 ppm ozone. The persistent nature of the ozone-induced MCM in rats documented in this report suggests that ozone exposure may have the potential to induce similar long-lasting alterations in the airways of humans.

Mucin gene (MUC 2 and MUC 5AC) upregulation by Gram-positive and Gram-negative bacteria.

Bacterial infection of the lung is associated with mucin overproduction. In partial explanation of this phenomenon, we recently reported that supernatant from the Gram-negative organism Pseudomonas (P.) aeruginosa contained an activity that upregulated transcription of the MUC 2 mucin gene [J.-D. Li, A. Dohrman, M. Gallup, S. Miyata, J. Gum, Y. Kim, J. Nadel, A. Prince, C. Basbaum, Transcriptional activation of mucin by P. aeruginosa lipopolysaccharide in the pathogenesis of cystic fibrosis lung disease, Proc. Natl. Acad. Sci. U.S.A., 94 (1997) 967-972]. The purpose of the present study was to determine whether mucin genes other than MUC 2 are so regulated and whether Gram-positive organisms also contain mucin stimulatory activity. Results from in situ hybridization and RNase protection assays showed that P. aeruginosa upregulates MUC 5AC as well as MUC 2 in both bronchial explants and cultured airway epithelial cells. The upregulation of both genes by P. aeruginosa can be mimicked by lipopolysaccharide (LPS) and can be blocked by the tyrosine kinase inhibitor genistein. In addition, both genes are upregulated by a variety of Gram-positive as well as Gram-negative organisms showing the same rank order of potency. These data indicate the existence of a general mechanism by which epithelial cells respond to the presence of bacteria by increasing mucin synthesis.

Activation of NF-kappaB via a Src-dependent Ras-MAPK-pp90rsk pathway is required for Pseudomonas aeruginosa-induced mucin overproduction in epithelial cells.

Cystic fibrosis (CF) is an autosomal recessive disorder, the most common lethal genetic disease in Caucasians. Respiratory disease is the major cause of morbidity and mortality. Indeed, 95% of CF patients die of respiratory failure. Pseudomonas aeruginosa, an opportunistic pathogen, chronically infects the lungs of over 85% of CF patients. It is ineradicable by antibiotics and responsible for airway mucus overproduction that contributes to airway obstruction and death. The molecular mechanisms underlying this pathology are unknown. Here we show that P. aeruginosa activates a c-Src-Ras-MEK1/2-MAPK-pp90rsk signaling pathway that leads to activation of nuclear factor NF-kappaB (p65/p50). Activated NF-kappaB binds to a kappaB site in the 5'-flanking region of the MUC2 gene and activates MUC2 mucin transcription. These studies bring new insight into bacterial-epithelial interactions and more specifically into the molecular pathogenesis of cystic fibrosis. Understanding these signaling and gene regulatory mechanisms opens up new therapeutic targets for cystic fibrosis.

Cloning of the amino-terminal and 5'-flanking region of the human MUC5AC mucin gene and transcriptional up-regulation by bacterial exoproducts.

To obtain gene regulatory sequence for the mucin gene MUC5AC, we have isolated the MUC5AC amino terminus cDNA and 5'-flanking region. This was possible through the use of rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) in which the 5' sequence of the human gastric mucin cDNA HGM-1 (1) was used to design the first MUC5AC-specific primer. Primers for subsequent rounds of RACE were designed from the 5'-ends of amplified RACE products. After five rounds of RACE-PCR, we could no longer generate upstream extensions of the cDNA and hypothesized that we had reached the 5'-end. Primer extension and RNase protection analysis confirmed this. Combined nucleotide sequence for the RACE-PCR products was 3.3 kb with an open reading frame encoding 1100 amino acids. A putative translation start site was found at nucleotide +48. This was followed by a 45 nucleotide putative signal sequence. This amino-terminal sequence contains no tandem repeats but is >60% similar to the amino-terminal nucleotide sequence of MUC2. The positions of cysteine residues in this MUC2-similar region are almost 100% conserved between the two genes. Northern analysis showed expression of cognate RNA in the stomach and airway but not muscle and esophagus. This pattern was the same as that obtained using previously reported 3'-MUC5AC sequences. We have cloned approximately 4 kb of genomic DNA upstream of the transcription start site and have sequenced 1366 nucleotides containing a TATA box, a CACCC box, and putative binding sites for NFkappaB and Sp 1. Within 4 kb of the transcription start site are elements mediating transcriptional up-regulation in response to bacterial exoproducts.

Sp1 protein contributes to airway-specific rat MUC 2 mucin gene transcription.

We have shown increases in the abundance of airway mucin mRNA during the pathogenesis of chronic obstructive pulmonary disease in rat models (Jany et al., 1991) and now seek to determine the underlying mechanisms. As transcriptional modulation may be involved, we provide here a functional analysis of the 5' flanking region of a rat mucin gene (MUC 2). Using deletion mutants to bp -859, we constructed expression cassettes in CAT vectors and transfected them into two MUC 2-expressing cell lines, SPOC 1, a rat airway epithelial cell line and IEC-6, a rat intestinal epithelial cell line, and into one MUC 2 non-expressing cell line, FR, a rat skin fibroblast cell line. Results indicated that nucleotides -59 to -40 mediated high level expression in SPOC 1, but not in the other cells. Used as a probe in gel shift assays, fragment -59/-40 formed complexes of differing mobilities when incubated with nuclear protein extracts from the three cell types. Mutation of the putative Sp1 binding site in the probe sequence interfered with protein binding in all three cell types, but anti-Sp1 antibody supershifted a band formed only by airway cell extracts. A model of airway cell-specific MUC 2 transcription is proposed.

A novel role for murine IL-4 in vivo: induction of MUC5AC gene expression and mucin hypersecretion.

Mucus hypersecretion and plugging of lower respiratory tract airways contributes to the morbidity and mortality associated with asthma. Interleukin (IL)-4 plays a putative role in some forms of asthma. Thus, transgenic mice that overexpress murine IL-4 selectively within the lung were used to study the effect of IL-4 on mucus glycoprotein gene expression and mucin release. Histologic examination of lung sections from IL-4 mice revealed that nonciliated epithelial cells from conducting airways were hypertrophic, due at least in part to the accumulation of mucus glycoprotein. The cytoplasm of these cells stained positively for glycoproteins using mucicarmine, alcian blue (AB), and periodic acid-Schiff (PAS). Ciliated cells were also enlarged but did not show any mucin-specific staining. Inclusion granules typically found in nonciliated (Clara) cells of control mice were absent in the IL-4 transgenic mice. Northern blot analysis of total RNA from lung tissue revealed that the expression of the MUC5AC, but not MUC2, mucin gene was distinctly upgraded in IL-4 transgenic mice compared to transgene-negative controls. In addition, a 5- to 10-fold increase in AB- and PAS-positive material was found in lavage fluid from IL-4 overexpressing mice compared to transgene-negative controls. Thus, the overexpression of IL-4 locally within the lung enhances mucus glycoprotein synthesis by altering gene expression, results in the accumulation of mucus glycoprotein in nonciliated epithelial cells, and induces the release of mucus into the airway lumen. We therefore hypothesize that the overproduction of mucus seen in some patients with asthma may be a direct result of the action of IL-4 within the inflamed lung.

Transcriptional activation of mucin by Pseudomonas aeruginosa lipopolysaccharide in the pathogenesis of cystic fibrosis lung disease.

An unresolved question in cystic fibrosis (CF) research is how mutations of the CF transmembrane conductance regulator, a Cl ion channel, cause airway mucus obstruction leading to fatal lung disease. Recent evidence has linked the CF transmembrane conductance regulator mutation to the onset and persistence of Pseudomonas aeruginosa infection in the airways, and here we provide evidence directly linking P. aeruginosa infection to mucus overproduction. We show that P. aeruginosa lipopolysaccharide profoundly upregulates transcription of the mucin gene MUC 2 in epithelial cells via inducible enhancer elements and that this effect is blocked by the tyrosine kinase inhibitors genistein and tyr-phostin AG 126. These findings improve our understanding of CF pathogenesis and suggest that the attenuation of mucin production by lipopolysaccharide antagonists and tyrosine kinase inhibitors could reduce morbidity and mortality in this disease.

Transcriptional regulation of the lysozyme gene in airway gland serous cells.

Lysozyme is expressed in serous, but not mucous, cells of the tracheobronchial glands and thereby constitutes a marker of the serous cell lineage in these glands. To identify DNA regulatory elements and transcription factors mediating the commitment of progenitor cells to the serous cell lineage, we have characterized the regulatory activity and DNA-protein interactions of the 5'-flanking region of the bovine lysozyme gene lys 5a. Results obtained from these studies indicate that although approximately 94 bp of 5'flanking DNA are necessary for high level expression in transient transfection assays, an evolutionarily conserved promoter within 66 bp of the transcription start site is sufficient to confer serous cell-specific expression. Farther upstream, within 6.1 kb of the 5' flanking region, are 4 silencers. Analysis of the serous cell-specific lysozyme promoter by electrophoretic mobility shift assay (EMSA) revealed the presence of binding sites for 3 serous cell nuclear proteins, designated LSF1, LSF2 and LSF3. Binding of LSF2 and LSF3 was localized to a 20-mer subdomain (-50/-30) of the cell-specific promoter using binding competition assays. More accurate identification of the protein binding site(s) was achieved through the use of mutagenesis, which implicated the motif 5' AAGGAAT 3' (-46/-40) in both protein binding and serous cell-specific transcriptional activity. This motif has previously been identified as a binding site for ets protein transcription factors, suggesting that serous cell-specific regulation of lys 5a transcription is partly controlled by the binding of ets-like protein(s) to the motif 5'AGGAAGT3'.

Molecular cloning of the amino-terminal region of a rat MUC 2 mucin gene homologue. Evidence for expression in both intestine and airway.

To obtain cDNAs for analysis of mucin gene transcription in rat models of human disease, we screened a rat intestinal cDNA library in lambda ZAPII using an upstream non-tandem repeat cDNA fragment of the human MUC 2 gene (Gum, J., Hicks, J., Toribara, N., Rothe, E., Lagace, R., and Y., K. (1992) J. Biol. Chem. 267, 21375-21383). Three cDNAs, 1-1, 8-1, and 21-1, were isolated. A translation start site was found in cDNA 21-1. Combined nucleotide sequence for the three cDNAs contained an open reading frame spanning 4546 base pairs. This amino-terminal sequence contains a non-tandem repeat domain enriched in cysteine (1391 residues) followed by an irregular tandem repeat domain (122 residues). Identity with the human gene is about 80% in the non-tandem repeat domain and about 38% in the irregular tandem repeat domain. Primer extension and S1 nuclease protection analysis indicate a transcription start site at 28 base pairs upstream of translation initiation. Northern analysis showed expression of cognate RNA in the intestine and airway but not heart and spleen. The cDNAs have been used to isolate the gene promoter, the structure of which should yield clues to the regulation of mucin expression in rat models of human disease.

Multiple cDNA sequences of bovine tracheal lysozyme.

The principal role of lysozyme is to prevent bacterial invasion at body surfaces. We are interested in how lysozyme is regulated at the surface of the respiratory tract, where the serous gland cell is regarded as the primary cellular source of this enzyme. Since the cow genome contains at least 10 lysozyme-like genes, our objective was to determine which of them are expressed in the cow tracheal gland serous cell. By screening tracheal cDNA libraries with a probe constructed from the cDNA encoding stomach lysozyme 2, we obtained 3 lysozyme cDNAs: 5a (1023 base pairs (bp)), 7a (1060 bp), and 14d (1249 bp). cDNA 7a corresponds to a previously reported gene (showing sequence identity to the stomach 2 lysozyme gene), whereas cDNAs 5a and 14d correspond to lysozyme genes not previously reported. Northern blot analysis of cow tracheal RNA showed lysozyme mRNAs of three distinct lengths. Based on hybridization with probes specific for each cDNA, we determined that the longest transcript corresponded to cDNA 5a, the shortest to 7a, and the intermediate-length transcript to 14d. Cultured cow tracheal gland serous cell RNA, reverse transcribed and amplified by the polymerase chain reaction with primers common to all three cDNAs, yielded a product that hybridized to oligonucleotide probes specific for all three cDNAs but most strongly to that for 5a. These results indicate that multiple lysozyme mRNAs are expressed in the cow trachea and that the lysozyme encoded by cDNA 5a is the major form expressed in the tracheal gland serous cell. This serous cell lysozyme is predicted to differ importantly in structure from both 7a and 14d lysozymes, with an arginine:lysine ratio almost 10-fold higher. The sequence differences may underlie functional differences, including variable resistance to proteolysis and variable affinity for large polyanions (e.g. mucins) found in the respiratory tract lumen.

Characterization of a rat airway cDNA encoding a mucin-like protein.

The purpose of this study was to isolate airway mucin cDNAs for use in studies of mucin biosynthesis in rat models of human airway disease. To this end, we screened a rat airway cDNA library with the human intestinal mucin cDNA SMUC 41 and obtained 7 positive clones. Preliminary characterization of each of these led us to focus on the clone expressing the 390 bp cDNA RAM 7s. Evidence indicating that RAM 7s encodes part of a rat airway mucin gene is that RAM 7s: (a) hybridizes in plaque lifts to SMUC 41, (b) hybridizes in Northern blots to large, polydisperse transcripts, (c) has a sequence encoding threonine-rich tandem repeats and (d) shows appropriate tissue-specific expression of cognate mRNA. The repetitive peptide encoded by RAM 7s includes five copies of the consensus sequence TTTTIITI. Because this sequence is different from those reported for two cDNAs previously isolated from rat intestinal libraries, we tentatively conclude that RAM 7s encodes part of a previously unidentified rat mucin gene.

Mucin gene expression in rat airways following infection and irritation.

Airway mucus hypersecretion occurs in response to infection and irritation and poses an important and poorly understood clinical problem. In order to gain insight into its pathogenesis, we have focused on an mRNA encoding the major mucus glycoprotein, mucin. Northern blots showed that mucin mRNA was abundant in the intestine of specific pathogen free rats whereas it was undetectable in the airways of these rats until pathogen-free conditions were suspended and rats acquired Sendai (Parainfluenza I) virus infections. Airway mucin hybridization signals in rats that were both infected with Sendai virus and exposed to SO2 were more intense than those in rats with infection alone. These results suggest that pathogen-and irritant-induced hypersecretion may be partly controlled at the level of mucin mRNA.

Human bronchus and intestine express the same mucin gene.

The amino acid and sugar composition of mucins from various organs is similar but not identical. This could arise by one or more of the following: organ-specific processing of a single core protein, organ-specific splicing of a single mucin mRNA, or organ-specific expression of various mucin genes. To begin to investigate the source of this variability, we examined (a) immunological cross-reactivity and (b) cDNA cross-hybridization, among several mucin-secreting organs of the human body. Peptide-directed antibodies raised against both nondeglycosylated (LS) and deglycosylated (HFB) intestinal mucin strongly stained mucous cells in the bronchial epithelium and submucosal glands, indicating homology between mucins of the bronchus and intestine at the peptide level. By screening a bronchus cDNA library with an intestinal mucin cDNA, SMUC-41, we isolated a bronchus mucin cDNA, HAM-1. This cDNA is 96% homologous to the first repeat of SMUC-41. HAM-1 hybridized to restriction fragments of human genomic DNA identical to those hybridizing to SMUC-41 on Southern blots. SMUC-41 also hybridized to polydisperse transcripts in the bronchus, cervix, gall bladder, and mammary gland, indicating mucin homology among all these organs at the RNA level. We conclude that the bronchus and intestine express a common mucin gene, which is likely co-expressed by at least several other mucin-secreting organs.

Modification of mucin gene expression in the airways of rats exposed to sulfur dioxide.

The molecular mechanisms mediating mucous cell metaplasia and hypersecretion in the respiratory tract are unknown. Previous work suggests that mucous metaplasia requires the induction of mucin gene expression. We are investigating this possibility by monitoring steady state levels of mucin mRNA in a model of mucous cell metaplasia induced by SO2 exposure. Male Sprague Dawley rats were exposed to 400 ppm SO2 gas in air for 3 h per day, 5 days per week for 0,1,2, or 3 weeks. Sham controls were exposed to air under similar conditions. After 3 weeks, morphological changes were apparent in the epithelium of SO2 exposed rats at all levels from the trachea to the distal airways. The epithelial thickness increased, as well as the number and size of glands in the trachea. Epithelial mucous (goblet) cells increased from 0 to 4.5 per mm in the trachea, 0.2 to 6.2 per mm in the main stem bronchi, and 0.2 to 22.7 per mm in the distal airways (mean values obtained for 3-6 tissue blocks per airway level per condition). In parallel experiments, we used SMUC41, a 950 bp human intestinal cDNA to isolate a human airway cDNA, HAM-1 from a cDNA library constructed in bacteriophage from human bronchial poly A+RNA. HAM-1 is a 90 bp cDNA encoding a threonine- and proline-rich peptide with 96% homology to the human intestinal cDNA SMUC-41. Next we probed total and poly A+ airway RNA from rats in each exposure condition with SMUC-41 or HAM-1. Blots were then stripped and reprobed with cDNA encoding beta actin. Densitometry values normalized for the amount of RNA loaded per lane (as determined by actin hybridization intensity) showed that mucin mRNA increased 8-9 fold as a function of SO2 exposure. This is consistent with the possibility that mucin gene transcription is induced by SO2 exposure, and may represent a primary event in the development of mucous metaplasia and hypersecretion.

The natural flavorprotein electron acceptor of trimethylamine dehydrogenase.

The isolation and partial characterization of a flavoprotein which functions as the electron acceptor of trimethylamine dehydrogenase (EC 1.5.99.7) from a methylotrophic bacterium is described. It has a molecular weight of 77,000 and is composed of two dissimilar subunits. All preparations examined contained only 1 mol of FAD/mol of the flavoprotein. Trimethylamine dehydrogenase, in the presence of trimethylamine or dithionite, reduced the flavoprotein to a stable anionic semiquinone form. No evidence for the participation of the fully reduced flavoprotein in catalysis could be obtained.