Herb pair of Ephedrae Herba-Armeniacae Semen Amarum alleviates airway injury in asthmatic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Ephedrae Herba (EH, Ephedra sinica Stapf.) and Armeniacae Semen Amarum (ASA, Prunus armeniaca L. var. ansu Maxim.) have been used to treat asthma, cold, fever, and cough in China for thousands of years. AIM OF THE STUDY: In this study, we aimed to investigate the optimal ratio of EH and ASA compatibility (EAC) to reduce airway injury in asthmatic rats and its possible mechanism. METHODS: Rats were sensitized with a mixture of acetylcholine chloride and histamine bisphosphate 1 h before sensitization by intragastric administration of EAC or dexamethasone or saline for 7 days. Subsequently, the ultrastructure of rat airway epithelial tissue changes, apoptosis of the airway epithelial cells, and the expression of mRNA and protein of EGRF and Bcl-2 were detected. RESULTS: Transmission electron microscope: EAC (groups C and E) had the most prominent effect on repairing airway epithelial cells' ultrastructural changes in asthmatic rats. TUNEL: dexamethasone and EAC (groups B、C、E and F) inhibited the apoptosis of airway epithelial cells in asthmatic rats (P < 0.05). In situ hybridization: EAC (group E) inhibited the overexpression of EGFR and Bcl-2 mRNA (P < 0.05).Western Blotting: EAC (groups A、B、C、E and F) inhibited the upregulation of airway epithelial EGFR and Bcl-2 protein expression (P < 0.01). CONCLUSIONS: Our findings indicate that EAC can inhibit abnormal changes in airway epithelial structure and apoptosis of airway epithelial cells, thereby alleviating airway injury. In this study, the best combination of EH and ASA to alleviate airway epithelial injury in asthmatic rats was group E (EH: ASA = 8: 4.5).

MiR-196-5p involvement in selenium deficiency-induced immune damage via targeting of NFκBIA in the chicken trachea.

Dietary selenium (Se) deficiency can induce multifarious immune injury in tissues, accompanied by inflammation and a decreased expression of selenoproteins. The results of previous studies indicated that these issues are associated with Se-mediated microRNAs involved in immune regulation, although the specific mechanisms associated with these interactions have not been reported in the trachea of chickens. To explore the effects of Se deficiency in the trachea of chickens and the role of miR-196-5p, we established correlational models of tracheal injury in chickens. One hundred broilers were divided into four groups, including a control group (C group), a Se deficient group (L group), a lipopolysaccharide (LPS)-induced control group (C + LPS group) and a LPS-induced Se deficient group (L + LPS group). Light microscopy observations indicated that the infiltration of inflammatory cells was the major histopathological change caused by Se deficiency. Furthermore, ultrastructural observation of the tracheal epithelium and ciliary showed typical inflammatory signs owing to Se deficiency. We determined the targeting relationship between miR-196-5p and NFκBIA by bioinformatics analysis. In the case of Se deficiency, the changes were detected as follows: 19 selenoproteins showed different degrees of decrease (p < 0.05). Significant inhibition of both antimicrobial peptides and immunoglobulin production were observed (p < 0.05). IκB-α (NFκBIA) expression degraded with the increasing miR-196-5p (p < 0.05), and the NF-κB pathway was activated. Thereafter, we can see a significant increase in the mRNA levels of inflammatory cytokines-related genes (tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin E (PTGE), interleukin (IL)-1ß, IL-6) and protein expression of NF-κB/iNOS pathway-related genes (NF-κB, iNOS, TNF-α, COX-2) (p < 0.05). The release of IL-2, interferon (IFN)-γ inhibited (p < 0.05) and the secretion of IL-4, IL-6 increased, suggesting the imbalance of Th1/Th2 (Th, helper T cell) cytokines. Compared to the control, the mRNA and protein expression levels of the anti-inflammatory system components with antioxidant activity (PPAR-γ/HO-1) were in an inhibitory state (p < 0.05). Antioxidases (SOD, CAT, GSH-Px) activities were suppressed. The activities of the peroxide markers (MDA, H2O2) were enhanced (p < 0.05). In addition, Se deficiency had a positive effect on the pathological changes of inflammation and the exceptional immunity in LPS-treated groups (p < 0.05). The results confirmed the relationship between miR-196-5p and NFκBIA in chickens, revealing that Se deficiency causes respiratory mucosal immune dysfunction via the miR-196-5p-NFκBIA axis, oxidative stress and inflammation. Moreover, Se deficiency exacerbates the inflammatory damage stimulated by LPS. Our work provides a theoretical basis for the prevention of tracheal injury owing to Se deficiency and can be used as a reference for comparative medicine. Furthermore, the targeted regulation of miR-196-5p and NFκBIA may contribute to the protection of the tracheal mucosa in chickens.

Toluene diisocyanate exposure induces laryngo-tracheal eosinophilia, which can be ameliorated by supplementation with antioxidant vitamins in guinea pigs.

OBJECTIVE: Toluene diisocyanate (TDI)-induced asthma is a common cause of occupational lung disease. In addition, a sore throat is one of the complaints of TDI-exposed workers. The aim of the present study was to determine whether TDI exposure induces laryngeal and/or tracheal lesions in experimental animals. MATERIAL AND METHODS: Guinea pigs underwent naris application of TDI three times, and their respiratory tracts were then examined using light and electron microscopy. Some animals simultaneously received vitamins C and E. which function as antioxidant agents. RESULTS: When TDI-treated animals showed the clinical sign of labored breathing, many eosinophils had appeared in the lamina propria and mucosa of both the larynx and trachea, which finally infiltrated the tract lumen through the ruptured epithelium. Laryngo-tracheal inflammation was more severe than that observed in the lungs. However, supplementation with antioxidant vitamins in TDI-treated animals ameliorated the respiratory eosinophilia. CONCLUSION: Naris application of TDI induced laryngotracheitis. which was significantly suppressed by the antioxidant vitamins, This implies a preventive effect of the vitamins on this occupational disease.

Air exposure causes oxidative stress in cultured bovine tracheal epithelial cells and produces a change in cellular glutathione systems.

An air-liquid interface culture system accelerates cell differentiation and growth in airway epithelial cells. In this study, the authors investigated whether glutathione (GSH) affects cell growth in an air-liquid interface culture. Various components of the cellular GSH system were measured and the number of cells was counted after conversion from an immersed feeding to an air-liquid interface. The effect of medium supplementation with cystine, a precursor of GSH, on cell growth was also examined. After conversion to an air-liquid interface, the cellular GSH level increased by 4 to 5-fold. The increase in GSH preceded the rise in cell number. The glutathione disulfide (GSSG)/total GSH ratio (an indicator of oxidative stress) increased initially and reached a plateau (time 0: 0.379%+/-0.054%; day 2: 0.824%+/-0.063%; day 6: 0.806%+/-0.093%). The level of glutathione reductase activity in the cells increased in a time-dependent manner, whereas the glutathione peroxidase activity level declined. When the amount of cystine in the medium was varied after conversion, the cellular GSH level was closely correlated with the rate of cell growth. In conclusion, air exposure causes oxidative stress in cultured cells and produces a change in the cellular glutathione system, resulting in the promotion of cell growth.

Lactosylated poly-L-lysine targets a potential lactose receptor in cystic fibrosis and non-cystic fibrosis airway epithelial cells.

Poly-L-lysine with 40% of the epsilon -amino groups substituted with lactosyl residues facilitated the internalization of lactosylated poly-L-lysine/cDNA complexes into cystic fibrosis (CF) and non-CF airway epithelial cells. It was previously shown that lactosylated poly-L-lysine enhanced the transfer of cDNA into the cell nucleus, resulting in transfection. The cell entry of lactosylated poly-L-lysine/cDNA complexes, however, has not been elucidated and we hypothesized that entry of the complex was by receptor-mediated endocytosis. It is shown here that binding of the vector/cDNA complexes to the cell membrane was inhibited by lactose but not N-acetyl glucosamine. Examination by electron microscopy revealed the complexes in clathrin-coated pits. Furthermore, the complexes colocalized with transferrin during cell entry and were shown in early endosomes. These results demonstrated that lactosylated poly-L-lysine/cDNA complexes enter airway epithelial cells via receptor-mediated endocytosis utilizing lactose-binding receptors, which employ the clathrin-coated pit for internalization. Taken together with the fact that nuclear translocation also is enhanced by lactose, these results demonstrate why lactosylated poly-L-lysine is an excellent vector for transfection of airway epithelial cells. Moreover, other carbohydrates covalently linked to poly-L-lysine for targeting other specific cell types, combined with lactosyl residues, can be designed for the development of other molecular conjugates for gene transfer.

Use of oolong tea extract staining of soft-tissue specimens in low-vacuum scanning electron microscope with a cooling stage.

For direct observation of the surface structures of soft-tissue specimens, we examined rat tracheal tissue in a low-vacuum scanning electron microscope (SEM) equipped with a cooling stage. In specimens fixed with glutaraldehyde and osmium tetroxide, back-scattered electron images of the surface structure could not be clearly observed in the low-vacuum SEM because of the disruption of fine structures and a low signal-to-noise (S/N) ratio. Processing of the specimens in 70% ethanol resulted in marked shrinkage, in contrast to results when processing in 30% ethanol. To overcome these problems, the trachea was initially fixed in 2.5% glutaraldehyde (0.1 M phosphate buffer pH 7.4), treated with a mixture of 0.2% oolong tea extract (OTE) and 2.5% glutaraldehyde, and postfixed in 1% osmium tetroxide. The sample was immersed in 30% ethanol and examined in a chilled SEM at -10 degrees C. The luminal coutour of the tracheal epithelial cells was clearly observed because of the decrease in shrinkage. Cilia of ciliated cells and microvilli of nonciliated cells were also clearly observed. These specimens also showed a high S/N ratio, thus allowing the observation of samples without the need for complete dehydration, critical-point drying, or metal coating. This OTE-incorporated conductive staining method is simple and rapid, and should prove to be highly useful for rapid SEM analyses of biological specimens.

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.

Inspired anaesthetic gas humidification improves thermal uniformity during canine whole body hyperthermia.

Supplying warmed saturated water vapour in anaesthetic gases during whole body hyperthermia (WBH) could potentially improve thermal uniformity in the trachea and esophagus. Four normal dogs were anaesthetized for WBH at 42 degrees C. A Puritan Bennett Cascade humidifier was used to supply anaesthetic gases saturated with water vapour at an average airway temperature of either 42 degrees C or 38 degrees C. Esophageal temperature was monitored at the thoracic inlet and 5 cm cephalad. Thermal dose was estimated by calculating equivalent minutes for an isoeffect at 43 degrees C (CEM 43 degrees Tx, where Tx is the site of temperature measurement). Endotracheal mucociliary transport velocity (MCTV) was determined before and 48 h following WBH by 99mTc-MAA scintigraphy. Compared to the 38 degrees C humidified gas group, dogs receiving 42 degrees C humidified gas reached 42 degrees C faster (p = 0.02) and had CEM 43 degrees T(esophageal) values equivalent to the target CEM 43 degrees T(rectal). Endotracheal MCTV with 42 degrees C humidified gas, however, was reduced 53% from baseline 48 h following WBH (p = 0.02). With 38 degrees C humidified gas, endotracheal mucociliary transport velocity was unchanged from baseline 48 h post WBH. Tracheal histology was examined using light and electron microscopy in four additional dogs euthanatized following 90 min of 42 degrees C humidified gas combined with WBH. There was no histological evidence of tracheal or lung thermal damage with 42 degrees C humidified gas in these four dogs. However, a moderate increase in tracheal goblet cell secretory granule staining was observed. This change could imply temporary heat-induced ciliary microtubule dysfunction, rather than decreased mucus production, as the likely mechanism of reduced mucociliary transport velocity 48 h following WBH. Administration of 42 degrees C humidified anaesthetic gases with WBH improves heating rate and esophageal thermal uniformity but temporarily depresses tracheal mucociliary transport velocity.

Preparation of cultured airway smooth muscle for study of intracellular element concentrations by X-ray microanalysis: comparison of whole cells with cryosections.

Methods for growing and preparing smooth muscle cells, isolated from rabbit trachealis, for X-ray microanalysis studies are presented. The cells are grown on Pioloform-covered gold grids supported on Thermanox coverslips. This provides a growth-compatible substrate which is easy to handle and is easily incorporated into routine cell culture studies. The cells are analysed as whole mounts after removal of growth medium by washing, followed by cryofixation and freeze drying. The effects of different washing media (0.3 M sucrose, 0.15 M ammonium acetate and distilled water) on cytoplasmic elemental content are discussed. A method for growing the cells as monolayers and mounting the cryofixed monolayers for cryosectioning is also given. Comparison of elemental concentrations in the cytoplasm of distilled-water washed cells with those of the cytoplasm of cryosectioned cells obtained from the same animal showed good agreement between values obtained from the two preparative procedures. These methods are therefore easily applied to the study of changes in intracellular element concentrations which may be important in understanding the mechanisms of proliferation which lead to increased airway smooth muscle mass in persistent severe asthma.

Quantitative ultrastructural analysis of the relationship between cell growth, shape change, and mucosecretory differentiation in cultured hamster tracheal epithelial cells exposed to retinoic acid.

Hamster tracheal epithelial cells in extended (32 degrees C) primary culture with and without supplemental retinoic acid (RA) were studied during the proliferative (5 days) and differentiation phases (11 days) by correlative transmission electron microscopy (EM) and light microscopic (LM) autoradiography to quantify the relationship between cell proliferation, shape change, and mucin granule expression. In retinyl acetate-containing control medium, cell numerical density was higher and [3H]thymidine labeling index (LI) lower at day 11 compared with day 5. The addition of 10(-7) M RA to the medium caused an increase in cell numerical density at both times. LI was increased by RA at 5 days and decreased at 11 days. Measurements of cell shape in ultrathin sections adjacent to LM autoradiographs made in the vertical plane demonstrated an RA-induced change from flat to cuboidal at 5 days and a more columnar phenotype at 11 days. Cells containing mucin granules were of two main types based on their ultrastructure. One type, seen at 5 and 11 days, contained diminutive mucin granules and had an LI of 50% at 11 days. Its LI and frequency (26%) were unaltered by RA. The other type, less frequent (15%) and present only at 11 days, was more columnar and contained mucous granules similar to those found in vivo. RA doubled the frequency of this cell type but did not affect its LI (11%). Cells of this type with more than five mucin granules in EM profile did not incorporate thymidine. The data indicate that RA accelerates and enhances cell shape change toward a more cuboidal phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

[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.

Effect of retinol on fetal lamb tracheal epithelium, with and without epidermal growth factor. A model for the effect of retinol on the healing lung of human premature infants.

Twelve pairs of fetal lambs were used to test the hypothesis that the necrotizing tracheobronchitis followed by squamous metaplasia seen in premature infants who develop chronic bronchopulmonary dysplasia might be related to low retinol stores and might, therefore, be reversed by retinol supplementation. Epidermal growth factor (EGF) was used to model the growth factor stimulus initiated by chronic wounding of the airways, and retinol was used as a differentiator of proliferating cells stimulated by EGF. Saline-treated animals were used as controls, as were fetal lambs receiving retinol alone or EGF alone. The effects of EGF on tracheal and bronchial epithelium consisted of proliferation of basal and intermediate cells, necrosis and slough of lining ciliated and mucous-producing cells, followed by squamous metaplasia. In fetal lambs given retinol, plasma, liver and lung retinol levels rose and mucous producing cells were increased in number. In the presence of EGF plus retinol, differentiation of mucous-producing cells was accelerated. We believe that this fetal lamb model with low initial levels of retinol in plasma, liver and lung, treated with EGF may mimic human premature infants with chronic bronchopulmonary dysplasia, and that the addition of retinol in amounts sufficient to raise their tissue levels produces a more normal surface epithelium in conducting airways.

Effects of all-trans retinol and cigarette smoke condensate on hamster tracheal epithelium in organ culture. I. A cell proliferation study.

The effects of cigarette smoke condensate (CSC) and all-trans retinol on the cell proliferative activity of vitamin A-deprived hamster tracheal epithelium have been studied in vitamin A-deficient, serum-free, hormone-supplemented medium in organ culture. In the absence of retinol, CSC induced a dose-dependent increase in labeling index (LI) during 12 days of culture. The basal cells were more sensitive to CSC exposure than non-basal cells during the first 6 to 8 culture days. However, in squamous metaplastic foci developing after culture day 6, both basal and non-basal cells in the mid-part of the epithelium were labeled. Physiological concentrations of all-trans retinol stimulated the non-basal LI and inhibited the basal cell LI. Compared with dimethylsulfoxide (DMSO), all retinol concentrations used in the present study inhibited the basal cell LI at each time point examined (4-12 days culture). Exposure of tracheal rings to retinol, either before or after exposure to CSC, or simultaneous exposure to retinol and CSC, clearly decreased the CSC-induced basal cell proliferative activity depending on the retinol concentration used. It is concluded from the present study that squamous metaplasia induced by vitamin A-deficiency or by CSC originates mainly from basal cells and that for the maintenance of these lesions, both basal and non-basal cells play a role. Furthermore, all-trans retinol inhibited CSC-induced basal cell proliferation.

Effects of all-trans retinol and cigarette smoke condensate on hamster tracheal epithelium in organ culture. II. A histomorphological study.

The effects of all-trans retinol and cigarette smoke condensate (CSC) on tissue morphology and cellular differentiation were investigated in vitamin A-deprived tracheal epithelium cultured in vitamin A-and serum-free hormone-supplemented medium. Physiological retinol concentrations prevented the development of hyperplasia and squamous metaplasia with or without keratinization, and induced differentiation to mucous cells. Squamous metaplastic foci with keratinization were observed during 12 days of culture with low retinol concentrations and with dimethylsulfoxide (DMSO) which was accompanied by an increased number of basal and indeterminate cells. CSC induced a dose-related hyperplasia and irregularly shaped foci of squamous metaplasia with atypical epithelial proliferation. In non-metaplastic epithelium, CSC exposure increased the number of ciliated cells. Hyperplasia and squamous metaplasia were inhibited if the tracheal rings were first treated with retinol followed by CSC exposure, or if the tracheas were simultaneously treated with retinol and CSC. CSC-exposure prior to retinol treatment induced similar histomorphological alterations as CSC alone.

Expression of tracheal differentiated functions in serum-free hormone-supplemented medium.

Most dissociated airway epithelial cells in culture express few of their in vivo functions and only to a limited degree. In this report, we demonstrate that hamster tracheal epithelial (HTE) cells cultured on a collagen gel substratum in a serum-free hormone-supplemented medium differentiate to cilia-beating and mucus-secreting cell types. The medium is Ham's F-12 supplemented with insulin, epidermal growth factor, transferrin, hydrocortisone, cholera toxin, bovine hypothalamus extract, and vitamin A. Under these culture conditions, HTE cells exhibit a growth rate of 24 h/population doubling and reach confluency, at a density of 2-5 X 10(4) cells/cm2, within 2 weeks. Both the collagen gel substratum and vitamin A of this culture system are important to the growth and differentiation of HTE cells in vitro. Evidence of HTE cell differentiation has been obtained at both the ultrastructural and the histochemical levels. In addition, a variety of biochemical studies (gel filtration, ion exchange column chromatography, enzyme digestion, nitrous acid treatment, and composition analysis) indicate the production of mucin-like glycoprotein in the HTE cultures. The levels of mucin-like glycoprotein were found to closely correlate with the histochemically quantitated levels of the mucous cell type. Kinetic studies demonstrate that HTE cells rapidly lose their differentiated features during the attachment stage of primary culture but redifferentiation occurs after the cultures reach confluency. The ability of HTE cells to grow and differentiate in this serum-free culture system in the absence of other cell types should greatly facilitate the study of mucociliary functions in vitro.

Tracheal ultrastructure in kerosene treated guinea pigs. A preliminary report.

p6high correlation between the usage of kerosene and the appearance of asthmatic crises has been demonstrated. The ultrastructure of the upper respiratory tract of animals treated with kerosene has not been previously reported. Kerosene aerosol was administered for 15 minutes daily during 21 days to adult male guinea pigs with fragments of trachea being processed for ultrathin electron microscopical studies. Controls did not receive any treatment. Trachea of guinea pigs submitted to kerosene aerosols showed swelling, ruffling and breakdown of the ciliary membrane. The regularly arranged ciliary border was disturbed to a certain degree in some areas by the development of cytoplasmatic protrusions at the apical part of the ciliated cells. An eosinophilic infiltrate was observed deep inside the epithelium and into the lamina propria. Therefore, these ciliary alterations can be considered as one of the most important changes induced by kerosene in tracheal epithelial cells. The protrusions may represent a sign of cell alteration produced by kerosene aerosol inhalation in the guinea pig.