The lactoperoxidase system functions in bacterial clearance of airways

Airway mucus is a complex mixture of secretory products that provides a multifaceted defense against pulmonary infection. Mucus contains antimicrobial peptides (e.g., defensins) and enzymes (e.g., lysozyme) although the contribution of these to airway sterility has not been tested in vivo. We have previously shown that an enzymatically active, heme-containing peroxidase comprises 1% of the soluble protein in sheep airway secretions, and it has been hypothesized that this airway peroxidase may function as a biocidal system. In this study, we show that sheep airway peroxidase is identical to milk lactoperoxidase (LPO) and that sheep airway secretions contain thiocyanate (SCN(-)) at concentrations necessary and sufficient for a functional peroxidase system that can protect against infection. We also show that airway LPO, like milk LPO, produces the biocidal compound hypothiocyanite (OSCN(-)) in vitro. Finally, we show that in vivo inhibition of airway LPO in sheep leads to a significant decrease in bacterial clearance from the airways. The data suggest that the LPO system is a major contributor to airway defenses. This discovery may have significant implications for chronic airway colonization seen in respiratory diseases such as cystic fibrosis.     

Enhancement of respiratory mucosal antiviral defenses by the oxidation of iodide

Recent reports postulate that the dual oxidase (DUOX) proteins function as part of a multicomponent oxidative pathway used by the respiratory mucosa to kill bacteria. The other components include epithelial ion transporters, which mediate the secretion of the oxidizable anion thiocyanate (SCN(-)) into airway surface liquid, and lactoperoxidase (LPO), which catalyzes the H(2)O(2)-dependent oxidation of the pseudohalide SCN(-) to yield the antimicrobial molecule hypothiocyanite (OSCN(-)). We hypothesized that this oxidative host defense system is also active against respiratory viruses. We evaluated the activity of oxidized LPO substrates against encapsidated and enveloped viruses. When tested for antiviral properties, the LPO-dependent production of OSCN(-) did not inactivate adenovirus or respiratory syncytial virus (RSV). However, substituting SCN(-) with the alternative LPO substrate iodide (I(-)) resulted in a marked reduction of both adenovirus transduction and RSV titer. Importantly, well-differentiated primary airway epithelia generated sufficient H(2)O(2) to inactivate adenovirus or RSV when LPO and I(-) were supplied. The administration of a single dose of 130 mg of oral potassium iodide to human subjects increased serum I(-) concentrations, and resulted in the accumulation of I(-) in upper airway secretions. These results suggest that the LPO/I(-)/H(2)O(2) system can contribute to airway antiviral defenses. Furthermore, the delivery of I(-) to the airway mucosa may augment innate antiviral immunity.     

Lysozyme secretion by submucosal glands protects the airway from bacterial infection

Submucosal glands are abundant (approximately 1 gland/mm2) secretory structures in the tracheobronchial airways of the human lung. Because submucosal glands express antibacterial proteins, it has been proposed that they contribute to lung defense. However, this concept is challenged by the fact that mice do not have submucosal glands in their bronchial airways, yet are quite resistant to bacterial lung infection. The contribution of airway submucosal glands to host defense is also debated as a pathophysiologic component of cystic fibrosis lung disease. Here, we asked whether submucosal glands protect airways against bacterial infection. By comparing tracheal xenograft airways with and without glands, we found that the presence of glands enhanced bacterial killing in vivo and by airway secretions in vitro. Moreover, immunodepletion studies suggested that lysozyme is a major antibacterial component secreted by submucosal glands. These studies provide evidence that submucosal glands are a major source of antibacterials critical for maintaining sterile airways.     

Activity of abundant antimicrobials of the human airway

Human airways produce several antimicrobial factors; the most abundant are lysozyme and lactoferrin. Despite their likely importance in preventing infection, and their possible key role in the pathogenesis of cystic fibrosis (CF), we know little about their antibacterial activity in the context of the CF airway. We found that abundant airway antimicrobial factors kill common CF pathogens, although Burkholderia was relatively resistant. To study the antibacterial activity, we developed a rapid, sensitive, and quantitative in vitro luminescence assay. Because NaCl concentrations may be elevated in CF airway surface liquid, we tested the effect of salt on antibacterial activity. Activity of individual factors and of airway lavage fluid was inhibited by high ionic strength, and it was particularly sensitive to divalent cations. However, it was not inhibited by nonionic osmolytes and thus did not require hypotonic liquid. The inhibition by ionic strength could be partially compensated by increased concentrations of antibacterial factors, thus there was no one unique salt concentration for inhibition. CF airway secretions also contain abundant mucin and elastase; however, these had no effect on antibacterial activity of lysozyme, lactoferrin, or airway lavage fluids. When studied at low NaCl concentrations, CF and non-CF airway lavage fluids contained similar levels of antibacterial activity. These results suggest approaches toward developing treatments aimed at preventing or reducing airway infections in individuals with CF.     

New models of the tracheal airway define the glandular contribution to airway surface fluid and electrolyte composition

Antibacterial defenses in the airway are dependent on multifactorial influences that determine the composition of both fluid and/or electrolytes at the surface of the airway and the secretory products that aid in bacterial killing and clearance. In cystic fibrosis (CF), these mechanisms of airway protection may be defective, leading to increased colonization with Pseudomonas aeruginosa. Submucosal glands, a predominant site of cystic fibrosis transmembrane conductance regulator (CFTR) protein expression in the airway, have been hypothesized to play an important role in protection of the airway. Furthermore, recent studies have suggested that the salt concentration at the airway surface may be a key factor in regulating the activity of antibacterial substances in the airway. To explore these issues, we have used a new model of the ferret tracheal airway to evaluate the contribution of submucosal glands in regulating airway surface fluid and electrolyte composition. Using tracheal xenograft models with and without submucosal glands, we have characterized several aspects of airway physiology that may be important in defining antibacterial properties. These endpoints included the contribution of submucosal glands in defining bioelectric properties of the surface airway epithelium, airway surface fluid (ASF) chloride composition, ASF volumes, and secretion of the antibacterial factor lysozyme. Findings from these studies demonstrate a significantly elevated secreted fluid volume (Vs) and chloride concentration ([Cl](s)) in ASF from airways with submucosal glands (Vs = 47 +/- 4 microl; [Cl](s) = 128 +/- 5 mM), as compared with xenograft airways without glands (Vs = 36 +/- 2 microl; [Cl](s) = 103 +/- 6 mM). Furthermore, a temperature labile factor secreted by submucosal glands appears to alter the baseline activation of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and/or diphenylamine-2-carboxylic acid-sensitive chloride channels in the surface airway epithelium. Lastly, the lysozyme content of tracheal airways with submucosal glands was 8.5-fold higher than were airways without glands. These studies demonstrate that submucosal glands affect both the ionic composition and bioelectric properties of the airway and suggest that models evaluating antibacterial properties of the airway in CF should take into account the contribution of glands in airway physiology.     

Increased carbon monoxide levels in the nasal airways of subjects with a history of seasonal allergic rhinitis and in patients with upper respiratory tract infection

BACKGROUND: Carbon monoxide (CO) has emerged as an endogenously produced gaseous mediator known to be involved in bronchial smooth muscle regulation. Increased amounts of CO have been found in exhaled air during asthma and lower airway inflammation. Recently CO has been shown to be produced in the nasal airways, but there are no reports of altered CO levels in nasal airways during inflammation. OBJECTIVE: This study was designed to investigate if CO levels increase in the human nasal airways during inflammatory conditions, such as allergy and upper airway respiratory tract infection (URTI). METHODS: CO was sampled separately from the upper and lower airways of 13 healthy control subjects, six patients with a history of allergic rhinitis and six patients with URTI. RESULTS: Nasal CO levels were increased in subjects with allergic rhinitis, compared to healthy controls (2.07 +/- 0.15 ppm, n = 6 and 1.62 +/- 0.08 ppm, n = 13, respectively, P < 0.01). CO levels were also increased in patients with URTI, compared to the same controls (1.92 +/- 0.09 ppm, n = 6, P < 0.05). Normal levels of CO were found in air from the lower airways among subjects with allergic rhinitis, whereas corresponding levels in the URTI patients were increased. CONCLUSION: The present data demonstrates that upper airway CO levels increase in parallel with different inflammatory stimuli, such as allergy and infection, suggesting a role for CO as marker or mediator of nasal inflammation.     

Purification and characterization of PLUNC from human tracheobronchial secretions

To study proteins secreted into the airway, we used secretions from primary human airway epithelial cells, re-differentiated at the air-liquid interface, and from patients intubated during surgery. A major protein of the cultured cell secretions was ethanol soluble. This protein was purified, analyzed by Edman degradation, matrix-assisted laser-desorption ionization time-of-flight mass spectroscopy of tryptic digests, and Western blots of two-dimensional electrophoresis gels using antisera against the purified preparation. The protein was identified as palate, lung, nasal epithelium clone protein (PLUNC). The protein had multiple truncated molecules, a pattern also seen in tracheal aspirates. PLUNC was poorly soluble in water (50 microg/ml) or in 50 mM NaCl but was more soluble in 75% ethanol (> 380 microg/ml). PLUNC secretion dramatically increased during the second week in air-liquid interface culture and continued to increase over time. Immunohistochemistry showed that PLUNC was expressed in human airway epithelium and submucosal glands. Although PLUNC is in the lipopolysaccharide (LPS)-binding protein (LBP) and bactericidal/permeability-increasing protein family of antibacterial host defense proteins, purified PLUNC failed to compete with LBP for the binding of LPS, whereas polymyxin B, a known inhibitor of LPS-LBP binding, did interfere with binding. This study showed that plunc gene product is expressed both in vivo and in vitro, detailed a method for its purification and provided basic information on its biochemical properties in secretions.     

Tracheobronchial epithelium of the sheep: IV. Lectin histochemical characterization of secretory epithelial cells

Conventional histochemical characterization of the mucus secretory apparatus is often difficult to reconcile with the biochemical analysis of respiratory secretions. This study was designed to examine the secretory glycoconjugates in airways using lectins with biochemically defined affinities for main sugar residues of mucus. We used five biotinylated lectins--DBA (Dolichos biflorus) and SBA (Glycine max) for N-acetyl galactosamine (galNAc), BSA I (Bandeiraea simplicifolia) and PNA (Arachis hypogea) for galactose (gal), and UEA I (Ulex europeus)--for detection of fucose (fuc) in HgCl2-fixed, paraffin-embedded, serially sectioned trachea, lobar and segmental bronchi and bronchioles of nine sheep. Lectins selectively localized the carbohydrate residues in luminal secretions, on epithelial cell surfaces, and in secretory cells. In proximal airways, the major carbohydrate residues in luminal secretions, cell surfaces, goblet cells, and glands were fuc and gal-NAc. PNA reacted mainly with apical granules of less than 10% of goblet cells, and gal residues were only detected in some of the mucous cells and on basolateral cell surfaces. Distal airways contained sparse secretion in the lumen, mucous cells contained weakly reactive fuc and gal-NAc, and the epithelial surfaces of Clara cells contained gal. Sugars abundant in the airway secretions were also the major component of cells in glands. We conclude that there is a correlation between specific sugar residues in secretory cells, glycocalyx, and luminal secretions in proximal and distal airways. This suggests that lectins may be used to obtain information about airway secretory cell composition from respiratory secretions.     

Innate Antimicrobial Activity of Nasal Secretions

Minimally manipulated nasal secretions, an accessible form of airway surface fluid, were tested against indigenous and added bacteria by using CFU assays. Antimicrobial activity was found to vary between donors and with different target bacteria and was markedly diminished by dilution of the airway secretions. Donor-to-donor differences in electrophoresis patterns of nasal secretions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and acid urea-PAGE analyses were readily observed, suggesting that polymorphic genes encode the secreted proteins. Three donors (of twenty-four total), whose nasal fluid yielded similar protein band patterns and did not kill indigenous bacteria, were determined to be heavy nasal carriers of Staphylococcus aureus. Their fluid was deficient in microbicidal activity toward a colonizing strain of S. aureus but the defect was corrected in vitro by a 1:1 addition of nasal fluid from noncarriers. The microbicidal activity of normal fluid was inactivated by heating it for 10 min to 100 degrees C and could not be restored solely by the addition of two major nasal antimicrobial proteins, lysozyme and lactoferrin. Several other known antimicrobial proteins and peptides, including statherin, secretory phospholipase A2, and defensins, were identified in nasal secretions and likely contribute to their total antimicrobial properties. Nasal fluid may serve as a useful model for the analysis of lower-airway secretions and their role in host defense against airway colonization and pulmonary infections.     

A method of obtaining, processing, and analyzing human intestinal secretions for antibody content

Human intestinal secretions can be readily obtained using a commercially available intestinal lavage solution. Although such secretions contained abundant protease activity, significant loss of immunoglobulins was prevented by the addition of a mixture of protease inhibitors. The total content of IgA, IgM, and IgG antibody in secretions was measured using sandwich ELISA. In the secretions of ten normal volunteers IgA was most abundant (197 micrograms/ml +/- 103 SD) followed by IgM (12.5 micrograms/ml +/- 6.8 SD) and IgG (0.24 micrograms/ml +/- 0.04 SD). The IgA in secretions was predominantly secretory IgA as shown by sucrose density centrifugation. The effect of intestinal secretions on the sensitivity of the antigen-specific ELISA was tested by adding murine myeloma IgA anti-TNP added to samples of human secretions. IgA anti-TNP activity could be detected as low as 1 ng/ml, and there was no evidence of interference with the ELISA by other constituents in the secretions. Using these methods an antigen-specific secretory IgA anti-cholera toxin B subunit response in the secretions of volunteers given an oral B subunit vaccine was readily demonstrated.     

SPLUNC1 promotes lung innate defense against Mycoplasma pneumoniae infection in mice

Short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is highly expressed in normal airways, but is dramatically decreased in allergic and cigarette smoke exposure settings. We have previously demonstrated SPLUNC1 in vitro antibacterial property against Mycoplasma pneumoniae (Mp). However, its in vivo biological functions remain unclear. The objectives of this study were to determine the in vivo functions of SPLUNC1 following bacterial (eg, Mp) infection, and to examine the underlying mechanisms. We generated SPLUNC1-deficient mice and utilized transgenic mice overexpressing human SPLUNC1 exclusively within the airway epithelium. These mice were infected with Mp and, twenty-four hours post infection, their host defense responses were compared to littermate controls. Mp levels and inflammatory cells increased in the lungs of SPLUNC1(-/-) mice as compared to wild type controls. SPLUNC1 deficiency was shown to contribute to impaired neutrophil activation. In contrast, mice overexpressing hSPLUNC1 exclusively in airway epithelial cells demonstrated lower Mp levels. Furthermore, neutrophil elastase activity was significantly increased in mice overexpressing hSPLUNC1. Lastly, we demonstrated that SPLUNC1 enhanced Mp-induced human neutrophil elastase (HNE) activity, and HNE directly inhibited the growth of Mp. Our findings demonstrate a critical in vivo role of SPLUNC1 in host defense against bacterial infection, and likely provide a novel therapeutic approach to restore impaired lung innate immune responses to bacteria in patients with chronic lung diseases.     

Biochemical evidence for an ecto alkaline phosphodiesterase I in human airways

Because dinucleotides are signaling molecules that can interact with cell surface receptors and regulate the rate of mucociliary clearance in lungs, we studied their metabolism by using human airway epithelial cells. A membrane-bound enzyme was detected on the mucosal surface of polarized epithelia that metabolized dinucleotides with a broad substrate specificity (diadenosine polyphosphates and diuridine polyphosphates [Up(n)U], n = 2 to 6). The enzymatic reaction yielded nucleoside monophosphates (NMP) and Np(n)(-)(1) (N = A or U), and was inhibited by nucleoside 5'-triphosphates (alpha,betamet adenosine triphosphate [ATP] > ATP >/= uridine triphosphate > guanidine triphosphate > cytidine triphosphate). The apparent Michaelis constant (K(m,app)) and apparent maximal velocity (V(max,app)) for [(3)H]Up(4)U were 22 +/- 4 microM and 0.24 +/- 0.05 nmoles. min(-)(1). cm(-)(2), respectively. Thymidine 5'-monophosphate p-nitrophenyl ester and adenosine diphosphate (ADP)- ribose, substrates of ecto alkaline phosphodiesterase I (PDE I) activities, were also hydrolyzed by the apical surface of airway epithelia. ADP-ribose competed with [(3)H]Up(4)U, with a K(i) of 23 +/- 3 microM. The metabolism of ADP-ribose and Ap(4)A was not affected by inhibitors of cyclic nucleotide phosphodiesterases (3-isobutyl-1-methylxanthine, Ro 20-1724, and 1,3-dipropyl-8-p-sulfophenylxanthine), but similarly inhibited by fluoride and N-ethylmaleimide. These results suggest that a PDE I is responsible for the hydrolysis of extracellular dinucleotides in human airways. The wide substrate specificity of PDE I suggests that it may be involved in several signaling events on the luminal surface of airway epithelia, including purinoceptor activation and cell surface protein ribosylation.     

Use of a monoclonal antibody enzyme-linked immunosorbent assay to measure human respiratory glycoprotein production in vitro

High-molecular-weight glycoprotein from human airway cultures was used to generate murine monoclonal antibodies, one of which recognizes a high-molecular-weight, hyaluronidase-resistant glycoprotein localized by immunofluorescent microscopy and immunogold electron microscopy to the secretory granules of human airway submucosal gland mucous cells and goblet cells. This monoclonal antibody was used to develop an enzyme-linked immunosorbent assay (ELISA) that was adapted to the study of respiratory glycoprotein secretion from human airways in vitro. Using the assay, the effect of a known mucus secretagogue, the cholinergic agonist methacholine, was studied on explant cultures of tissue from human bronchus or from human nasal mucosa. In studies of human bronchus explants, methacholine, 100 and 10 microM, stimulated increased secretion of respiratory glycoprotein (RGP) by 109 +/- 8% (n = 14; P less than 0.001) and 96 +/- 14% (n = 9; P less than 0.001), respectively, above control values. In studies of human nasal turbinate mucosal explants, methacholine, 100 and 10 microM, stimulated increased secretion of RGP by 75 +/- 28% (n = 7; P less than 0.01) and 70 +/- 21% (n = 4; P less than 0.01) above control values. An ELISA for the measurement of RGP secretion may provide a sensitive and more specific method for the performance of in vitro studies of RGP secretion from human tissues.     

Chronic obstructive pulmonary disease is associated with enhanced bronchial expression of FGF-1, FGF-2, and FGFR-1

An important feature of chronic obstructive pulmonary disease (COPD) is airway remodelling, the molecular mechanisms of which are poorly understood. In this study, the role of fibroblast growth factors (FGF-1 and FGF-2) and their receptor, FGFR-1, was assessed in bronchial airway wall remodelling in patients with COPD (FEV1 < 75%; n = 15) and without COPD (FEV1 > 85%; n = 16). FGF-1 and FGFR-1 were immunolocalized in bronchial epithelium, airway smooth muscle (ASM), submucosal glandular epithelium, and vascular smooth muscle. Quantitative digital image analysis revealed increased cytoplasmic expression of FGF-2 in bronchial epithelium (0.35 +/- 0.03 vs 0.20 +/- 0.04, p < 0.008) and nuclear localization in ASM (p < 0.0001) in COPD patients compared with controls. Elevated levels of FGFR-1 in ASM (p < 0.005) and of FGF-1 (p < 0.04) and FGFR-1 (p < 0.001) in bronchial epithelium were observed. In cultured human ASM cells, FGF-1 and/or FGF-2 (10 ng/ml) induced cellular proliferation, as shown by [3H]thymidine incorporation and by cell number counts. Steady-state mRNA levels of FGFR-1 were elevated in human ASM cells treated with either FGF-1 or FGF-2. The increased bronchial expression of fibroblast growth factors and their receptor in patients with COPD, and the mitogenic response of human ASM cells to FGFs in vitro suggest a potential role for the FGF/FGFR-1 system in the remodelling of bronchial airways in COPD.     

Antibacterial activity of hydrogen peroxide and the lactoperoxidase-hydrogen peroxide-thiocyanate system against oral streptococci.

In secreted fluids, the enzyme lactoperoxidase (LP) catalyzes the oxidation of thiocyanate ion (SCN-) by hydrogen peroxide (H2O2), producing the weak oxidizing agent hypothiocyanite (OSCN-), which has bacteriostatic activity. However, H2O2 has antibacterial activity in the absence of LP and thiocyanate (SCN-). Therefore, LP may increase antibacterial activity by using H2O2 to produce a more effective inhibitor of bacterial metabolism and growth, or LP may protect bacteria against the toxicity of H2O2 by converting H2O2 to a less-potent oxidizing agent. To clarify the role of LP, the antibacterial activities of H2O2 and the LP-H2O2-SCN- system were compared by measuring loss of viability and inhibition of bacterial metabolism and growth. The relative toxicity of H2O2 and the LP system to oral streptococci was found to depend on the length of time that the bacteria were exposed to the agents. During incubations of up to 4 h, the LP system was from 10 to 500 times more effective than H2O2 as an inhibitor of glucose metabolism, lactic acid production, and growth. However, if no more H2O2 was added, the concentration of the inhibitor OSCN- fell because of slow decomposition of OSCN-, and when OSCN- fell below 0.01 mM, the bacteria resumed metabolism and growth. In contrast, the activity of H2O2 increased with time. H2O2 persisted in the medium for long periods of time because H2O2 reacted slowly with the bacteria and streptococci lack the enzyme catalase, which converts H2O2 to oxygen and water. After 24 h of exposure, H2O2 was as effective as the LP system as an inhibitor of metabolism. H2O2 also caused a time-dependent loss of viability, whereas the LP system had little bactericidal activity. The concentration of H2O2 required to kill half the bacteria within 15 s was 1.8 M (6%) but fell to 0.3 M (1%) at 2 min, to 10 mM (0.03%) at 1 h, and to 0.2 mM (0.0007%) with a 24-h exposure. The results indicate that if high levels of H2O2 can be sustained for long periods of time, H2O2 is an effective bactericidal agent, and the presence of LP and SCN- protects streptococci against killing by H2O2. Nevertheless, the combination of LP, H2O2, and SCN- is much more effective than H2O2 alone as an inhibitor of bacterial metabolism and growth.     

Defensins: key players or bystanders in infection, injury, and repair in the lung?

Antimicrobial peptides have been identified as key elements in the innate host defense against infection. Recent studies have indicated that the activity of antimicrobial peptides may be decreased in cystic fibrosis, suggesting a major role for these peptides in host defense against infection. One of the most intensively studied classes of antimicrobial peptides are defensins. Defensins comprise a family of cationic peptides that in human subjects can be divided into the alpha- and beta-defensin subfamilies. The alpha-defensins are produced by neutrophils and intestinal Paneth's cells, whereas beta-defensins are mainly produced by epithelial cells. Although studies on beta-defensins have so far focused on their antimicrobial activity, studies on alpha-defensins have suggested a role of these peptides in inflammation, wound repair, and specific immune responses. alpha-Defensins, which accumulate in airway secretions of patients with various chronic inflammatory lung disorders, were shown to be cytotoxic toward airway epithelial cells and to induce chemokine secretion in several cell types. Furthermore, the capacity of alpha-defensins to promote bacterial adherence to epithelial cells in vitro further supports a role for these peptides in the pathogenesis of chronic obstructive pulmonary disease and cystic fibrosis. Increased numbers of neutrophils are also present in the airways of patients with asthma, suggesting that neutrophils are involved in the pathogenesis of this disease. Because defensins are able to induce histamine release by mast cells and increase the airway hyperresponsiveness to histamine, it is tempting to speculate that defensins may also contribute to the inflammatory processes in asthma. Besides these proinflammatory effects, alpha-defensins may also display anti-inflammatory activities, including regulation of complement activation and proteinase inhibitor secretion. Finally, defensins may be involved in wound repair because defensins increase epithelial cell proliferation. Thus recent defensin research has revealed potential links between the innate and acquired immune system.     

Characteristics of improved NOVA magnesium ion-selective electrode: changes of ionized magnesium values and reference interval in healthy children.

Following the report of interference between the thiocyanate ion (SCN-) and NOVA's previous ion-selective electrode (ISE) for ionized magnesium (iMg2+), NOVA has developed a new ISE which eliminates the effect of SCN-. Two hundred and sixty healthy children were divided into two groups; those who had presented when using NOVA's previous ISE (group A; n = 160) and those using NOVA's new ISE (group B; n = 100). The mean iMg2+ value and the mean iMg2+ percent fraction (iMg2+/serum Mg) were significantly higher in group B than in group A (0.59 +/- 0.03 vs 0.54 +/- 0.03 mmol/L for iMg2+; p < 0.001 and 64.8 +/- 3.1 vs 58.2 +/- 4.1 per cent for iMg2+ percent fraction; p < 0.001). The mean serum SCN- level was 0.023 +/- 0.008 mmol/L in group A (n = 8) and 0.0.21 +/- 0.007 mmol/L in group B (n = 12), and was not significantly different between the two groups. The suspected change of iMg2+ value interfered by SCN- was 0.037 mmol/L in group A. The difference of iMg2+ percent fraction between two groups was higher at high serum magnesium (SMg) levels. The reference interval of iMg2+ was 0.56-0.62 mmo/L in healthy children with the NOVA's new ISE, and was constant irrespective of growth. The NOVA's previous iMg2+ ISE may be interfered with mainly by SCN-. The newly designed ISE eliminated these effects especially at higher SMg levels.     

Modification of gene expression of the small airway epithelium in response to cigarette smoking

The earliest morphologic evidence of changes in the airways associated with chronic cigarette smoking is in the small airways. To help understand how smoking modifies small airway structure and function, we developed a strategy using fiberoptic bronchoscopy and brushing to sample the human small airway (10th-12th order) bronchial epithelium to assess gene expression (Affymetrix HG-U133A and HG-133 Plus 2.0 array) in phenotypically normal smokers (n = 16, 25 +/- 7 pack-years) compared to matched nonsmokers (n = 17). Compared to samples from large (second to third order) bronchi, the small airway samples had a higher proportion of ciliated cells, but less basal, undifferentiated, and secretory cells, and contained Clara cells. Even though the smokers were phenotypically normal, microarray analysis of gene expression of the small airway epithelium of the smokers compared to the nonsmokers demonstrated up- and downregulation of genes in multiple categories relevant to the pathogenesis of chronic obstructive lung disease (COPD), including genes coding for cytokines/innate immunity, apoptosis, mucin, response to oxidants and xenobiotics, and general cellular processes. In the context that COPD starts in the small airways, these gene expression changes in the small airway epithelium in phenotypically normal smokers are candidates for the development of therapeutic strategies to prevent the onset of COPD.     

Validity and reproducibility of morphologic analysis of nasal secretions obtained using ultrasonic nebulization of hypertonic solution.

BACKGROUND: Collection of nasal secretions is important for the evaluation of upper airways inflammation in many nasal disorders. OBJECTIVE: To study the validity and reproducibility of nasal secretion cellularity induced by nebulization of hypertonic solution in patients with allergic rhinitis (AR), patients with nonallergic rhinitis with eosinophilia syndrome (NARES), and control subjects. METHODS: Sixty-eight individuals (29 with AR [mean +/- SD age, 33.3 +/- 16.9 years], 23 with NARES [mean +/- SD age, 46.4 +/- 16.6 years], and 16 controls [mean +/- SD age, 42.1 +/- 15.1 years]) underwent ultrasonic nebulization of hypertonic (4.5%) saline solution on 2 different occasions to study the validity and reproducibility of total and differential cell counts of nasal secretions. RESULTS: The mean +/- SD percentage of eosinophils was significantly higher in samples from patients with AR (20.8% +/- 23.1%) and NARES (18.7% +/- 22.8%) than in samples from controls (0.6% +/- 0.6%; P < .001 for both). There was a significant correlation between 2 samples of nasal secretions obtained on 2 different occasions for percentages of macrophages, neutrophils, eosinophils, and epithelial cells. CONCLUSIONS: The analysis of nasal secretions obtained using ultrasonic nebulization of hypertonic solution can distinguish patients with AR and NARES from controls. The reproducibility of this technique is good for macrophages, neutrophils, eosinophils, and epithelial cells. This method could be used to detect nasal airway inflammation in clinical settings.     

Antimicrobial activity of human cervical mucus

The antibacterial activity of human cervical mucus (CM) was examined on standardized microbial colonized agar plates (agar diffusion test). In parallel, the lysozyme content of CM was determined by means of a turbidimetric test system in aliquots of the same CM specimens. Suspensions of living lyophilized Micrococcus lysedeikticus were used as bacterial substrate. Testing was performed in a total of 133 CM samples, obtained at mid-cycle from sexually active women from unselected infertile couples with a median age of 30 (range 21-42) years. All mucus specimens showed considerable antibacterial activity with clearly visible circular inhibition zones around the CM-filled holes in the colonized agar plates. Related to the effect of hen's egg white (HEW)-lysozyme on the same plates, the median activity of the CM specimens in the agar diffusion test was equivalent to 33.0 (range 6.4-391.4) microg/ml HEW-lysozyme. However, there was a wide inter-individual range of antibacterial effects of cervical secretions. The cervical index did not significantly influence the outcome of either test. The pH of the endocervical CM also was not correlated with the antibacterial effect. Sexual activity leading to the presence of spermatozoa in CM considerably increased its antibacterial effect. The activity was markedly higher in samples obtained within hours after intercourse compared with those taken after sexual abstinence of >/=5 days (P < 0.05). In microbially colonized CM specimens compared to sterile CM, all obtained under hormonally standardized conditions, the antibacterial activity in the agar plate test was significantly lower (P < 0.05). The results of this pilot study demonstrate the considerable antibacterial activity of human CM.