Evaluation of HO-1-u-1 cell line as an in vitro model for sublingual drug delivery involving passive diffusion--Initial validation studies.

The aim of this study was to provide preliminary validation of a new sublingual mucosal cell line (HO-1-u-1) for use as in vitro sublingual drug delivery screening of compounds involving passive diffusion. HO-1-u-1 cells were seeded on cell culture inserts. The ultrastructure and integrity of cell layers, inter-passage variation and directionality of drug transport, and apparent permeability coefficient (P(app)) of eight beta-blockers (representing compounds involving passive diffusion) were determined. HO-1-u-1 cells grown on inserts formed stratified and epithelial-like structure and maintained the typical histological features of normal human sublingual epithelium. The maximal integrity of the cell layer was reached in 23 days. No significant inter-passage variation was found at the passages ranging from 2 to 11 when measured by radiolabeled transcellular and paracellular markers (testosterone and mannitol, respectively). Bidirectional transport studies confirmed the passive diffusion as the mechanism of transport for these markers. The P(app) of eight beta-blockers across HO-1-u-1 cell culture ranged from 2.89+/-0.17 to 6.37+/-0.37x10(-6)cm/s and correlated well to the P(app) obtained from porcine sublingual mucosa (r(2)=0.647 and 0.83 when excluding propranolol). The above results indicate that the HO-1-u-1 cells grown on inserts may offer as a potentially in vitro model for screening sublingual drug permeation involving passive diffusion.

[Efficacy of sorption therapy in patients with cicatricial esophageal stenosis].

We examined 110 patients treated conservatively for cicatricial esophageal stenosis including expansion on the string. The patients were divided into three groups: controls (n = 35), receiving adjuvant SUMS-1 (n = 38) and given adjuvant enterosgel (n = 37). According to electron microscopy, enterosorbents make esophageal mucosa denser by decreasing interstitial spaces as a result of microcirculatory improvement and reduction of edema. Enterosorbents elevate total protein and sugar in the blood.

Ultrastructural study of the alveolar lining and the bronchial mucus layer by block staining with oolong tea extract: the role of various surfactant materials.

To investigate the roles of various surfactant materials in the lung, we examined rat lung fixed with a mixture of 0.2% oolong tea extract (which contains various polyphenols) and 2.5% glutaraldehyde by electron microscopy. We also measured the surface tension of various isolated surfactant fractions, with a Wilhelm balance. A fraction containing lamellar bodies and a fraction containing lattice-like structures were obtained by discontinuous sucrose gradient centrifugation. From the 15 000 g supernatant, a fraction containing electron-dense amorphous materials was obtained as the 105 g precipitate. The fraction containing lamellar bodies and the fraction containing electron-dense amorphous materials displayed surfactant activity, but the fraction containing lattice-like structures did not. The lamellar bodies were found to be gradually secreted from type II epithelial cells while self-decomposition occurred. The alveolar lining layer had the form of a thin film consisting of electron-dense amorphous materials. These electron-dense amorphous materials may be precursors of the phospholipid film, which exhibits surfactant activity, on the alveolar surface. Lattice-like structures and lamellar bodies were found to be located in the interalveolar pores. The interalveolar pores were filled with surfactant, and this indicated that they do not play a role in the collateral ventilation of the alveoli. It may be that the lattice-like structures serve to connect lung epithelial cells in the interalveolar pores, as well as serving as the basis for the formation of the alveolar ducts. The bronchial mucus layer, which consisted of fibrillar mucous materials, was not divided into an epiphase and a hypophase. A surfactant, in the form of an osmiophilic surface film and some trilaminar materials, was found to cover the mucus layer. Thus, it is possible that the lamellar bodies could be transformed into various surfactant materials, which then serve bronchial or alveolar mechanical and physiological functions.

Effects of hydrogen peroxide on the guinea-pig tracheobronchial mucosa in vivo.

Lumenal entry of plasma (mucosal exudation) is a key feature of airway inflammation. In airways challenged with histamine-type mediators and allergen the mucosal exudation response occurs without causing epithelial derangement and without increased airway absorption. In contrast, reactive oxygen metabolites may cause mucosal damage. In this study, involving guinea-pig airways, we have examined effects of H2O2 on airway exudation and absorption in vivo. Vehicle or H2O2 (0.1 and 0.5 M) was superfused onto the tracheobronchial mucosal surface through an oro-tracheal catheter. 125I-albumin, given intravenously, was determined in tracheobronchial tissue and in lavage fluids 10 min after challenge as an index of mucosal exudation of plasma. The tracheobronchial mucosa was also examined by scanning electron microscopy. In separate animals, 99mTc-DTPA was superfused 20 min after vehicle or H2O2 (0.1 and 0.5 M) had been given. A gamma camera determined the disappearance rate of 99mTc-DTPA from the airways as an index of airway absorption. The high dose of H2O2 (0.5 M) produced epithelial damage, increased the absorption of 99mTc-DTPA (P < 0.001), and increased the exudation of plasma (P < 0.001). Notably, it appeared that all extravasated plasma had entered the airway lumen within 10 min. These data demonstrate that H2O2 differs from exudative autacoids such as histamine by causing both epithelial damage and plasma exudation responses. These data also agree with the view that the epithelial lining determines the rate of absorption and is responsible for the valve-like function that allows lumenal entry of extravasated bulk plasma without any increased inward perviousness.

X-ray microanalysis of native airway surface liquid collected by cryotechnique.

The airway surface liquid (ASL) that lines the surface epithelium of the tracheobronchial tree is of vital importance to the airway defence against microbial invasion and damage due to environmental factors. Little is known about the ASL collected in situ in native conditions, owing to difficulties in collecting ASL without causing damage to the airway mucosa. We have developed a method to collect and analyse the elemental composition of tracheal ASL in pathogen-free mice. A specially designed cryoprobe, adapted to the internal curvature of the mouse trachea, was used to collect the native ASL from the tracheal surface. The complete ASL elemental composition including [Na] = 5.5 +/- 0.3, [Cl] = 1.3 +/- 0.3, [K] = 1.1 +/- 0.2, [Ca] = 1.2 +/- 0.3, [P] = 1.5 +/- 0.8, [S] = 1.7 +/- 0.4 and [Mg] = 1.3 +/- 0.4 mmol L-1 was determined by X-ray micro-analysis. We demonstrate here that the technique that we used for ASL collection maintained perfectly the airway epithelial integrity and functionality.

Interaction of Haemophilus parasuis with nasal and tracheal mucosa following intranasal inoculation of cesarean derived colostrum deprived (CDCD) swine.

Twenty-three cesarean derived, colostrum deprived pigs were obtained at 5 wk of age and inoculated intranasally with either 1.4 x 10(8) colony forming units of Haemophilus parasuis or sterile phosphate buffered saline. Pigs were euthanized at 4, 8, 12, 18, 26, or 36 h post-inoculation and tissues from the oropharynx and respiratory tract were obtained for qualitative bacterial culture, immunohistochemistry for H. parasuis antigens, and light and transmission electron microscopy. Haemophilus parasuis was consistently isolated from the nasal cavity (17/17, 100%) and trachea (13/17, 76%) and rarely isolated from the lung (3/17, 18%) and blood stream (1/17, 6%) of infected pigs. Antigens of H. parasuis were sporadically detected on the nasal mucosa (6/17, 35%) and trachea (8/17, 47%). Light microscopic lesions included submucosal and intraepithelial infiltrates of neutrophils and infrequent, patchy loss of cilia. Ultrastructural changes in nasal mucosal epithelial cells included cell protrusion, loss of cilia, and dilation of the cytocavitary network. Bacteria were infrequently identified and were either within an amorphous material at the apical surface of the cilia or were between individual cilia. These results suggest H. parasuis associates with the nasal mucosa and can induce a suppurative rhinitis with nasal mucosal epithelial cell degeneration. This process may represent an initial event in the pathogenesis of H. parasuis infection of swine.

Haemophilus influenzae infection of human respiratory mucosa in low concentrations of antibiotics.

We examined the effects of 0.25 and 0.5 minimal inhibitory concentrations (MIC) of amoxicillin, loracarbef, and ciprofloxacin on the interaction of a clinical isolate of nontypable Haemophilus influenzae (NTHi) with human adenoid organ culture. Adenoid tissue was embedded in agar so that only the mucosal surface was exposed. Minimum essential medium containing NTHi with or without antibiotics was added to the organ culture and incubated with 5% CO2 at 37 degrees C for 24 h. The organ cultures (n = 6) were assessed for several parameters by light microscopy (LM) and transmission electron microscopy (TEM). Bacterial viable counts after 24 h were not significantly different in all organ cultures. Compared with uninfected controls at 24 h, infection with NTHi caused significant (p < 0.05) damage to epithelium as assessed by LM: reduced ciliary beat frequency (CBF), disruption of epithelium integrity, and reduced number of ciliated sites. TEM showed extrusion of cells from the epithelial surface, loss of cilia from ciliated cells, cytoplasmic blebbing, and mitochondrial damage. In the presence of 0.25 and 0.5 MIC of all three antibiotics, the mucosal damage was significantly less (p < 0.05). We conclude that in the presence of sub-MIC levels of amoxicillin, loracarbef, and ciprofloxacin, NTHi infection causes less functional (CBF) and structural damage.

[Experimental study of modified yu ping feng powder on antibacterial adhesion of tracheal mucosa in mice model of chronic bronchitis].

In order to observe the influence of modified Yu Ping Feng San (MYPFS) on bacterial adhesion of tracheal mucosa, four experiments of bacterial adhesion in pneumatic tract were conducted, in which mice of chronic bronchitis model (CBM) induced by SO2 stimulation and another health control group breathed in aerosol contained Pseudomonas aeruginosa under the same condition were observed. The results showed that, with scanning electron microscopy, ultrastructural lesions on tracheal mucosa surface and adhesive bacterial number in CBM administrated MYPFS were far less than that in CBM without MYPFS (P < 0.001), and quantitative culture of Pseudomonas aeruginosa with tracheal tissue homogenate was also markedly reduced. However, the tracheal mucosa of healthy control animals were intact, the adhesive bacteria were not found. It is suggested that bacterial adhesion was closely related to the injury of tracheal-mucosa, and MYPFS could play a role of anti-bacterial adhesion through the protection of tracheal mucosa epithelium or reduction of pneumatic tract injury. These were quite in accordance with the theories of traditional Chinese medicine in "strengthening body resistance to eliminate the pathogenic factor", so that they provided experimental evidence for TCM tonics to prevent and treat infection of respiratory tract.

[Effect of jia wei yubingfengsan on bacterial adhesion to mouse tracheain chronic bronchitis model].

The experiment was designed to test the effect of Jia Wei Yubingfengsan (JW-YBFS) in chronic bronchitis of mice model. Two groups of mice model was used, one of which received JW-YBFS by oral administration another received only water as control experiment. The accurate quantification of bacterial adhesiveness by culture method showed that in JW-YBFS receiving mice viable counts of bacteria is much less than the control mice. In addition pathological examination showing the injury of tracheate mucosal epithelium was not found in JW-YBFS receiving mice while control mice showing typical chronic bronchitis lesion. The results demonstrated that JW-YBFS plays significantly role in bronchitis mice model. Our experimental data are in good agreement with Chinese medical conclusion, which means Fu Zheng Gu Ben.

Effects of cadmium acetate and sodium selenite on mucociliary functions and adenosine triphosphate content in mouse trachea organ cultures.

The effects of cadmium acetate and sodium selenite in mouse trachea organ culture have been studied separately and in combination. Ciliary activity, morphology, rate of total protein and glycoconjugate (i.e. glycoprotein and proteoglycan) synthesis/secretion and ATP content were investigated. Exposure to 10 microM cadmium acetate or 2000 microM sodium selenite resulted in a complete cessation of ciliary activity within 5 h. With cadmium acetate also a swelling of epithelial cells was observed. Sodium selenite (250-2000 microM) delayed by 2-3 h the inhibitory effect of cadmium acetate (5-20 microM) on ciliary activity. The rate of protein synthesis, as determined by incorporation of [3H]proline, was reduced by 13% and 44% at exposure for 4 h at 37 degrees C to 250 microM and 500 microM sodium selenite respectively, the effect being partly abolished by cadmium acetate. With 5 microM and 10 microM cadmium acetate the rate of glycoconjugate synthesis, as measured by incorporation of [3H]glucosamine, increased by 50% and 69%, respectively, after incubation for 4 h. This increase was partly reduced by sodium selenite. Neither cadmium acetate nor sodium selenite had any effect on the rate of total protein or glycoconjugate secretion. The ATP content in trachea rings was reduced by 48% and 54% after incubation for 4 h with 250 microM and 500 microM sodium selenite, respectively. No significant effect of cadmium acetate was found on ATP content. An antagonistic effect of sodium selenite and cadmium acetate in mouse trachea organ culture is suggested from the present experiments.