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An accurate mathematical model of transmucosal gas exchange is prerequisite to understanding middle ear (ME) physiology. Current models require experimentally measured gas species time constants for all extant conditions as input parameters. However, studies on pulmonary gas exchange have shown that a morphometric model that incorporates more fundamental physiochemical and anatomic parameters accurately simulates transport from which the species time constants can be derived for all extant conditions. Here, we implemented a variant of that model for ME gas exchange that requires the measurement of diffusional length (tau) for the ME mucosa. That measure contributes to the mucosal diffusing capacity and reflects the resistance to gas flow between air space and capillary. Two methods for measuring tau have been proposed: linear distance between the air-mucosal boundary and capillary and the harmonic mean of all contributing pathway lengths. Oxygen diffusing capacity was calculated for different ME mucosal geometries by using the two tau measures, and the results were compared with those predicted by a detailed, two-dimensional finite element analysis. Predictive accuracy was improved by incorporating the harmonic tau measure, which captures important information regarding variations in capillary shape and distribution. However, compared with the oxygen diffusing capacity derived from the finite element analysis, both measures yielded nonlinear, positively biased estimates. The morphometric techniques underestimate diffusion length by failing to account for the curvilinear gas flow pathways predicted by the finite element model.

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Phosphatidylcholine (PC) is the main phospholipid in lung surfactant and, more specifically, dipalmitoyl PC (PC16:0/16:0) is the major surface-active component. Several studies have tentatively shown that eustachian tube lavage fluid (ETLF) contains surface-active material. The aim of the present study was to determine, using electrospray ionization mass spectrometry, whether the phospholipid molecular species composition of ETLF is similar to that of lung surfactant. PC was the main component of both ETLF and bronchoalveolar lavage fluid (BALF). The concentration of phosphatidylethanolamine was higher and phosphatidylglycerol was undetectable in ETLF compared with BALF. The molecular species composition of PC in ETLF was notably different from that of BALF, palmitoyloleoyl PC being the major component. Importantly, given its predominance in BALF PC, the concentration of PC16:0/16:0 was low in ETLF. As expected on the basis of this molecular species composition of PC, ETLF did not generate low surface tension values under dynamic compression in a pulsating bubble surfactometer. We conclude that the surfactant in ET is different from lung surfactant, and that low surface tension is not a major determinant of ETLF function.

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Six different lectin probes were used to examine alterations of the cell surface carbohydrates in the chinchilla eustachian tube (ET) lumen subsequent to the intranasal (i.n.) challenge with the Streptococcus pneumoniae parent strain, D39, or its isogenic derivative, DeltaNA1, which is deficient in neuraminidase NanA. The labelling pattern revealed that the binding of wheat germ agglutinin (WGA), Erythrina cristagalli lectin (ECL), peanut agglutinin (PNA), Bandeiraea simplicifolia lectin II (BSL II) and succinylated wheat germ agglutinin (SWGA) were increased in the lumenal surface of the ET in the D39 inoculated cohort compared to the uninfected control, which indicated that N-acetylglucosamine (GlcNAc) and D-galactose residues were exposed. Concurrently, decreased labelling with Sambucus nigra agglutinin (SNA) indicated that there were few sialic acid residues remaining in the ET epithelium subsequent to i.n. inoculation with D39. The DeltaNA1 neuraminidase deficient mutant, however, did not induce any significant changes in the lectin labelling patterns, and was comparable to that of the control cohort. We propose that products of the nanA gene have a significant impact on the changes of the carbohydrate moieties in the ET epithelium and may be responsible for the previously reported increased ability of the D39 parent to colonize the nasopharynx and invade the middle ear.

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RNA analysis is essential for understanding biological activities of a cell or tissue. Unfortunately, retrieval of RNA from existing archives of human temporal bones has proven extremely difficult due to degradation of RNA molecules. The major factors that contribute to degradation of RNA in specimens from autopsied temporal bones are tissue autolysis due to time elapsed before autopsy, and technical problems in processing the bones after harvest. We therefore focused on improving the survival of RNA in human temporal bones by shortening the time to autopsy and through modification of the processing technique by removing targeted tissues directly from the temporal bones and by avoiding time-consuming decalcification and celloidin-embedding. Eight temporal bones collected at immediate autopsies were used in this study. Representative mRNAs, ranging from high (MUC5B, physically unstable) to low (beta-actin, physically stable) molecular weights, and from abundant (MUC5B) to non-abundant (MUC1) RNA, were studied by in situ hybridization, Northern blot technique, or both. Using this modified protocol in autopsies performed up to 6 h after death, the existence of mRNAs was demonstrated in all bones studied. This improved method demonstrates the feasibility of the use of autopsied temporal bone tissues for RNA analysis.

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The literature proving the presence of a surface tension lowering substance (STLS) on the lining layer of mammalian Eustachian tube (ET) is critically reviewed. A further review of the chemical studies on tubal washings based on chromatographic analysis methods (TLC and HPLC) is performed, and is concluded that ET epithelium is coated by a mixture of phospholipids, similar but not identical to the pulmonary surfactant and with similar but less powerful surface activity. In both cases, and with minor differences between the different mammalian species, phosphatidylcholine (PC), and in particular its disaturated fraction, dipalmitoilphosphatidylcholine (DPPC), is the predominating and the most active compound. ET surfactant is synthesized by ET epithelium and secreted in form of osmiophilic multilamellar bodies into the tubal lumen. The exact function of the ET surfactant is not fully understood: it may play an important role in ET physiology by facilitating the tubal opening to allow for aeration of the middle ear and adequate drainage or could act as a release agent, preventing solid-to-solid adhesion of the tubal walls and contrasting the adhesive action of the glycoproteins of the mucous blanket. On the other hand a phospholipidic surfactant seems to be produced by the mucosa of the other parts of the upper airways, i.e. nose and trachea. In this case a surface active agent could act in preventing the transudation of serum into the lumen, in enhancing the phagocytosis or in facilitating the mucociliary transport. Recent data on humans, suggesting that a relative deficiency or an alterated production of tubal surfactant could play a role in the pathogenesis of secretory otitis media (SOM) or middle ear effusion (MEE), are reviewed. Administration of exogenous surfactant or pharmacological stimulation of the production of tubal surfactant could improve ET function and be of value in some cases of SOM. Personal data, suggesting than ambroxol (a drug stimulating the production of pulmonary surfactant by the alveolar type II pneumocytes) exerts a similar activating effect on the tubotympanal secretory cells, are reported. These data support the results of clinical studies on the treatment of SOM with ambroxol.

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The distribution of secretory cells and surfactant-like lamellar bodies in mucosa of the guinea pig eustachian tube were studied ultracytochemically. Classified by means of their morphologic characteristics, three types of secretory cells were identified. The dark granulated cells were predominant in the tympanic orifice, the mixed cells were predominant in the isthmus portion, while the light cells were predominant in the pharyngeal orifice. The distribution of the secretory cells in different part of the eustachian tube might play a role in the pathogenesis of middle ear effusions. Surfactant-like lamellar bodies were found in ciliated cells, nonciliated cells and secretory cells of the eustachian tube mucosa. Surfactant-like lamellar bodies (surfactant-like substance) are important in maintaining the physiological function of the eustachian tube, their behavior in normal and pathological states should be further studied.

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The changes in the cell surface carbohydrates of the eustachian tube (ET) and middle ear subsequent to the intranasal (i.n.) inoculation of Streptococcus pneumoniae (Spn) type 6A were studied in the chinchilla model of otitis media (OM) using a lectin histochemical technique with six different lectins (SNA, WGA, Succ WGA, BSL II, PNA, ECL). The labeling pattern revealed not only the removal of the terminal sialic acid, but also exposure of N-acetylglucosamine (GlcNAc), a component of the trisaccharide receptor for Spn previously identified on human pharyngeal cells. The removal of sialic acid residues progressed from the nasopharyngeal to the tympanic orifice and was most pronounced in those animals from which Spn could be isolated from the middle ear. Our data indicate an alteration of the normal lectin labeling pattern and exposure of GlcNAc restricted mainly to the roof and neck portion, along the course of the eustachian tube. Exposure of part or all of a Spn adherence receptor structure by the pneumococcal enzymes, may facilitate colonization, invasion of the middle ear, and induction of OM.

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The guttural pouch is a large, air-filled diverticulum of the auditory tube, present in the horse and other species. Lipid analysis of saline lavage from the equine guttural pouch has demonstrated the presence of phospholipids and neutral lipids in amounts that are variable but consistently greater than in any other species described. A stain specific for choline-containing phospholipids has demonstrated the presence of phospholipid-containing vesicles only within the cells of subepithelial, seromucoidlike glands, suggesting that these cells incorporate phospholipids in their secretions. The functional significance of surface-active agents in the guttural pouch may be different from that proposed for other species because of the unique anatomical design and the different proposed functions of the guttural pouch.

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To examine the role of surface tension lowering substances (STLSs), we measured changes in passive opening pressure (OP) and closing pressure (CP) in eustachian tubes of guinea pigs before and after washing the tubes with various solutions (saline solution, artificial pulmonary surfactant, synthetic phospholipids, and detergent)). The percent decreases in OP and CP in tubes washed with artificial surfactant were significantly higher than those washed with saline solution. Those washed with synthetic phospholipids or detergent (Triton X-100) did not differ from those washed with saline solution. In guinea pigs with experimental otitis media produced by inoculation of formalin-killed Haemophilus influenzae into the middle ear cavity, the percent decreases in OP and CP in tubes washed with artificial pulmonary surfactant were significantly higher than in those washed with saline solution. Artificial pulmonary surfactant was injected into the middle ear cavity of the guinea pigs with otitis media. At 5 days after inoculation, the inflammatory changes of the middle ear were much milder in animals with intratemporal application of artificial pulmonary surfactant than in those with saline application. These results suggest the possible efficacy of treatment with artificial pulmonary surfactant for otitis media.

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In this study, neuraminidase (sialidase) and 6 different lectins, wheat germ agglutinin (WGA), Limax flavus agglutinin (LFA), Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), peanut agglutinin (PNA), and Amaranthin, were used to histochemically characterize the carbohydrate structure of glycoconjugate in the murine eustachian tube pharyngeal orifice. A microwave irradiation technique was used to reduce the incubation time and background staining. After neuraminidase treatment, WGA, LFA, MAA, Amaranthin, and PNA stained epithelial goblet cells, glandular mucous cells, cell surfaces, and the mucous blanket. Without neuraminidase treatment, SNA and PNA did not stain any secretory cells. These results revealed that sialoglycoconjugates in the murine eustachian tube pharyngeal orifice are produced from epithelial goblet cells and glandular mucous cells, are present on cell surfaces and within the mucous blanket, and that their terminal trisaccharide linkage appears to be the sequence Neu5Ac (alpha 2-3) Gal (beta 1-3) GalNAc.

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The effect of ambroxol administration on phospholipid and phosphatidyl-choline contents of rabbit eustachian tube and lung washings and on [14C]-choline incorporation by rabbit eustachian tube and lung tissue has been studied. Despite minor differences, the drug exerts the same activating effect in both locations. The results add a further piece of evidence to the several similarities existing between the lung surfactant and the surface-active substances present on the eustachian tube.

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Various biotinylated lectins were used to characterize and semiquantitate glycoconjugate residues in the tubotympanum. Epithelial goblet cells were stained predominantly by WGA, LFA, SNA, RCA-I, Con-A, LCA, SBA, PHA-E, and UEA; this finding suggests they contain alpha-neuraminic acid, beta-galactose, alpha-mannose, N-acetyl alpha-galactosamine, and alpha-fucose. Glandular mucous cells were stained predominantly by WGA, LFA, SNA, and RCA-I; this finding suggests that they contain alpha-neuraminic acid and beta-galactose. The glandular serous cells were stained predominantly by Con-A, WGA, and LFA; this finding suggests that they produced alpha-mannose and alpha-neuraminic acid that represented serum-type glycoprotein. The positive staining of epithelial goblet cells and glandular mucous cells with PNA after neuraminidase digestion suggests that they produced mucin-type glycoproteins. The staining of the mucous blanket by WGA, LFA, SNA, RCA-I, LCA, PNA, SBA, PHA-E, and UEA suggests the presence of alpha-neuraminic acid, beta-galactose, N-acetyl alpha-galactosamine, and alpha-fucose. The epithelial cell (nonsecretory) surface was stained largely by WGA, LFA, SNA, RCA-I, Con-A, and LCA; this finding suggests the presence of alpha-neuraminic acid, beta-galactose, and alpha-mannose.

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In this work an appropriate high-performance liquid chromatography method was set up to guarantee specificity, sensitivity, precision and accuracy in analyzing dipalmitoylphosphatidylcholine (DPPC) in rabbit eustachian tube washings, as well as to determine its varying levels after administration of ambroxol chloride. The procedure is based on a post-column derivatization with fluorescence detection using 1,6-diphenyl-1,3,5-hexatriene which exhibits increased fluorescence in a lipid environment. DPPC was chromatographed on a Hypersil C18. The mobile phase for the isocratic elution consisted of 40 mmol/l choline chloride in methanol-tetrahydrofuran (97:3). Ambroxol was given to a group of New Zealand white rabbits at a dose of 30 mg/kg. A second group receiving vehicle only acted as controls.

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The present study was conducted to characterize and localize the glycoconjugates in the tubotympanum (auditory or eustachian tube and middle ear cavity) of chinchilla on an ultrastructural level, using lectin-gold complexes with six different lectins: BPA, ConA, RCA-1, WGA, LFA, and SNA. A comparison of the affinity of these lectins demonstrated the heterogeneity of secretory cells. The glandular serous cells and epithelial dark granulated cells produced "serum"-type glycoprotein. The glandular mucous cells and goblet cells produced dominantly "mucin"-type glycoprotein in the light granules, but "serum"-type glycoprotein in the dark cores. The labeling of LFA and SNA showed that sialic acids existed mainly in the mucinous granules of secretory cells and ciliated epithelium glycocalyx, and in the mucous blanket. The results also suggested that the dominant linkage of sialic acids of mucin is a Neu5Ac(alpha 2-6)Gal/GalNAc sequence. Furthermore, the data obtained from ConA and BPA suggested that initial O-glycosylation of mucin took place in the cis side of the Golgi apparatus and that initial N-glycosylation of the serum occurred in the rough endoplasmic reticulum.

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It has been demonstrated that the eustachian tube and middle ear epithelium produce Tubal Surface Active Substances (TSAS), which facilitate the opening of the eustachian tube. In order to characterize the biochemical contents of chinchilla TSAS, the tubal washings were analyzed using 2-D thin layer chromatography. The results indicate that phosphatidylcholine was the predominant phospholipid, followed by sphingomyelin, phosphatidylinositol phosphatidylethanolamine, and phosphatidylserine. In comparison, pulmonary lavage showed phosphatidylcholine to be highest allowed by phosphatidylethanolamine and sphingomyelin. Phosphatidylcholine/phosphatidylethanolanim ratios were 5:1 in the tubal lavage, and 8:1 in the pulmonary lavage. Phosphatidylcholine/sphingomyelin ratios were 2:1 in the tubal lavage, and 67:1 in the pulmonary lavage. It is concluded that the biochemical content of TSAS is similar but not identical to that of pulmonary surfactants.

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