Dosing intact birch pollen grains at the air-liquid interface (ALI) to the immortalized human bronchial epithelial cell line BEAS-2B.

In real life, humans are exposed to whole pollen grains at the air epithelial barrier. We developed a system for in vitro dosing of whole pollen grains at the Air-Liquid Interface (ALI) and studied their effect on the immortalized human bronchial epithelial cell line BEAS-2B. Pollen are sticky and large particles. Dosing pollen needs resuspension of single particles rather than clusters, and subsequent transportation to the cells with little loss to the walls of the instrumentation i.e. in a straight line. To avoid high speed impacting insults to cells we chose sedimentation by gravity as a delivery step. Pollen was resuspended into single particles by pressured air. A pollen dispersion unit including PTFE coating of the walls and reduced air pressure limited impaction loss to the walls. The loss of pollen to the system was still about 40%. A linear dose effect curve resulted in 327-2834 pollen/cm2 (± 6.1%), the latter concentration being calculated as the amount deposited on epithelial cells on high pollen days. After whole pollen exposure, the largest differential gene expression at the transcriptomic level was late, about 7 hours after exposure. Inflammatory and response to stimulus related genes were up-regulated. We developed a whole pollen exposure air-liquid interface system (Pollen-ALI), in which cells can be gently and reliably dosed.

Attenuating effect of H+K+ATPase inhibitors on airway cough hypersensitivity induced by allergic airway inflammation in guinea-pigs.

BACKGROUND: Gastrooesophageal reflux (GER) is a frequent cause of chronic cough. Several investigators have indicated that inhibitors of H(+)K(+)ATPase (proton pump inhibitors; PPIs) could relieve coughing via inhibition of acid reflux. However, we considered that PPIs might directly inhibit increased cough reflex sensitivity. OBJECTIVE: The present study was designed to examine whether PPIs directly inhibit antigen-induced increase in cough reflex sensitivity and to elucidate the mechanism. METHODS: Actively sensitized guinea-pigs were challenged with aerosol antigen (ovalbumin, OVA) and cough reflex sensitivity to inhaled capsaicin was measured 24 h later. The PPIs (omeprazole and rabeprazole) or the histamine H(2) blocker cimetidine were administered intraperitoneally 1 h before OVA challenge and before measuring cough reflex sensitivity, then bronchoalveolar lavage fluid (BALF) was immediately collected. The pH of the fluid obtained by bronchial washing was determined after examining the effect of rabeprazole on the cough response to capsaicin. RESULTS: The number of coughs elicited by capsaicin was significantly increased 24 h after challenge with OVA compared with saline, indicating antigen-induced increase in cough reflex sensitivity. Both PPIs dose dependently and significantly inhibited antigen-induced cough hypersensitivity. Omeprazole did not influence the antigen-induced increase in the total number of cells or ratio (%) of eosinophils in BALF. Cimetidine did not affect the antigen-induced cough hypersensitivity or cellular components of BALF. The pH of the bronchial washing fluid was significantly decreased in antigen-challenged animals. Rabeprazole did not affect the antigen-induced decrease in the pH of bronchial washing fluid. CONCLUSION: These findings show that PPIs, but not histamine H(2) blockers, can directly decrease antigen-induced cough reflex hypersensitivity, while the mechanism remains unclear.

[Function of MMP/TIMP on airway remodeling of bronchial asthma and treating effects of Zhichuan capsule].

OBJECTIVE: To explore the mechanism of the bronchial asthma and to study the treating effects of Zhichuan Capsule on the airway remodeling of asthmatic model rats. METHODS: The rat model was established by being sensitized and activated with different density of ovalbumin through prolonged and repeated exposure for 8 weeks. The rats were randomly divided into model group, Zhichuan Capsule treated group, dexameson treated group, and Zhichuan Capsule and dexameson treated group. Another group of normal rats were taken as control. General histological changes were observed by hematoxylin and eosin stained sections. Being standardized by internal perimeter (Pi), the wall thickness (d), internal area (Ai), outer area (Ao) and wall area (WA) of the airway were quantified by computer-assisted image analysis system. The express of MMP-9, TIMP-1, Col I, Col III and ColV in the airway were examined by immunocytochemical methods. During the course of airway remodeling, the dynamic changes of model rats were observed at different time points (2, 4, 6 and 8 weeks after the activating). Statistical comparison was performed by ANOVA followed by Fisher LSD test. RESULTS: (1) Histologic examination showed eosinophil infiltration within the airway walls, epithelial damage, excessive mucus in the lumen and edema in the submucosa of the airways in model rats, and that the collagen deposition increased accompanied by increasing of TIMP-1. In the model rats, MMP-9 increased at the time point of 2 weeks, but it decreased in the late stage (8 weeks after activating) of airway remodeling. And the level of TIMP-1 was far higher than MMP-9 at the time point of 8 weeks. (2) Zhichuan Capsule could down-regulate the level of TIMP-1 in the airway wall, as well as the thickness of airway wall and the collagen deposition. And there were progressing effects when it was used together with dexameson. CONCLUSION: (1) The early increase of MMP-9 is a key point to start remodeling; and the increase of TIMP-1 in the late stage, which inhibits collagenase activity, may play an important role in developing airway fibrosis. Imbalance between MMP-9 and TIMP-1 is a marker of airway remodeling. (2) Zhichuan Capsule can decrease the deposition of collagen and suppress the airway remodeling by inhibiting the TIMP-1 expression.

Ascorbic acid in nasal and tracheobronchial airway lining fluids.

Ascorbic acid (AA) is thought to be an important antioxidant in the respiratory tract, whose regulation is yet to be fully characterized. We investigated whether AA in respiratory tract lining fluids (RTLFs) can be augmented by oral supplementation with AA. Plasma, nasal lavage fluids (NLFs), induced sputum (IS), and saliva were analyzed for AA immediately before and 2 h after ingestion of 2 g of AA in 13 healthy subjects. Concentrations of AA (median and range) were 52.5 (16.0-88.5), 2.4 (0.18-4.66), 2.4 (0.18-6.00), and 0.55 (0.18-18.90) micromol/l, respectively. Two hours after ingestion of AA, plasma AA increased 2-fold (p = .004), NLF AA increased 3-fold (p = .039), but IS and saliva AA did not increase. As AA concentrations in saliva and tracheobronchial secretions were low compared with other common extracellular components (such as urate), we evaluated the fate of AA in these fluids. Addition of AA to freshly obtained saliva or IS resulted in rapid depletion, which could be largely prevented or reversed by sodium azide or dithiothreitol. These findings suggest that oxidant-producing systems in saliva and airway secretions, such as heme peroxidases and other oxidizing substances, rapidly consume AA. Whereas oral supplementation resulted in detectable increases of AA in NLFs, its levels in tracheobronchial lining fluid, as measured by IS, were unaffected and remained relatively low, suggesting that AA may play a less significant antioxidant role in this compartment as compared with most other extracellular compartments.

Long-term inhalation of high-dose nitric oxide increases intraalveolar activation of coagulation system in mice.

Inhalation of nitric oxide (NO) is useful for the treatment of patients with pulmonary hypertension. However, the potential toxicity of inhaled NO is still unclear. Coagulation activation plays an important role in lung injury. We assessed the effect of low- and high-dose inhaled NO on the coagulation system in the intraalveolar space of mice. The animals were assigned to five groups (n = 6): [RA] group, mice exposed to fresh air alone; [RA+2 ppm NO] group, fresh air and 2 ppm NO; [RA+40 ppm NO] group, fresh air and 40 ppm NO; [RA+2 ppm NO+O(2)] group, fresh air, 2 ppm NO and O(2); and [RA+40 ppm NO+O(2)] group, fresh air, 40 ppm NO and O(2). Each group was treated for 3 wk. Lung specimens of [RA+40 ppm NO] and [RA+40 ppm NO+O(2)] groups showed significant nitrotyrosine immunoreactivity. BALF concentrations of total protein, thrombin and soluble tissue factor were significantly increased in mice of [RA+40 ppm NO] and [RA+40 ppm NO+O(2)] groups compared with [RA] group. However, BALF concentrations of total protein, thrombin, and soluble tissue factor were not significantly increased in mice of [RA+2 ppm NO] and [RA+2 ppm NO+O(2)] groups compared with [RA] group. Lung tissue factor mRNA expression was higher in the high-dose NO group than in the low-dose NO group. NO donor increased significantly tissue factor activity on alveolar epithelial cells. This study has shown for the first time that long-term inhalation of high, but not low, concentration of NO may activate the clotting system by increasing the lung expression of tissue factor.

Pathogenesis of infection induced by an adenovirus isolated from a goat.

OBJECTIVE: To determine the pathogenic potential of an adenovirus isolated from a goat. ANIMALS: 14 colostrum-deprived, isolation-reared goat kids approximately 3 weeks old. PROCEDURE: Kids were inoculated with either cell culture fluid containing adenovirus (n = 10) or uninfected cell culture fluid (n = 4): 2 ml transtracheally and 1 ml/nostril. Clinical signs of disease and rectal temperature were recorded daily; nasal secretion and fecal specimens were collected daily. Control kids were necropsied, 2/d, on postinoculation days (PID) 5 and 10. Virus-inoculated kids were necropsied on PID 3, 5, 7, 10, and 28. After necropsy, lung, liver, kidney, and brain specimens were aseptically collected for virus isolation attempts. Tracheal fluid was collected on sterile cotton swabs. Turbinate, trachea, lung, mediastinal lymph node, liver, kidney, duodenum, jejunum, ileum, mesenteric lymph node, colon, and brain specimens were collected for histologic evaluation. RESULTS: Kids developed mild-to-moderate clinical respiratory tract infection. Virus was recovered consistently from nasal secretion and sporadically from fecal specimens. Grossly, there were multiple areas of atelectasis and hyperemia, principally in the cranioventral portion of the lungs. Microscopically, there was detachment and sloughing of foci of epithelial cells of the terminal bronchioles and alveoli. In kids necropsied late in the disease, these changes were accompanied by hyperplasia of type-II epithelial cells. Viral inclusions were not an obvious feature, but a few cells contained probable inclusions. CONCLUSIONS AND CLINICAL RELEVANCE: The caprine adenovirus reported here is capable of inducing respiratory tract disease and lesions in the lungs of young kids.

Characterization of the human mucin gene MUC5AC: a consensus cysteine-rich domain for 11p15 mucin genes?

To date five human mucin cDNAs (MUC2, 5A, 5B, 5C and 6) mapped to 11p15.3-15.5, so it appears that this chromosome region might contain several distinct gene loci for mucins. Three of these cDNAs, MUC5A, B and C, were cloned in our laboratory and previously published. A common number, 5, was recommended by the Human Gene Mapping Nomenclature Committee to designate them because of their common provenance from human tracheobronchial mucosa. In order to define whether they are products of the same gene locus or distinct loci, we describe in this paper physical mapping of these cDNAs using the strategy of analysis of CpG islands by pulse-field gel electrophoresis. The data suggest that MUC5A and MUC5C are part of the same gene (called MUC5AC) which is distinct from MUC5B. In the second part of this work, complete sequences of the inserts corresponding to previously described (JER47, JER58) and novel (JER62, JUL32, MAR2, MAR10 and MAR11) cDNAs of the so-called MUC5AC gene are presented and analysed. The data show that in this mucin gene, the tandem repeat domain is interrupted several times with a subdomain encoding a 130 amino acid cysteine-rich peptide in which the TR3A and TR3B peptides previously isolated by Rose et al. [Rose, Kaufman and Martin (1989) J. Biol. Chem., 264, 8193-8199] from airway mucins are found. A consensus peptide sequence for these subdomains involving invariant positions of most of the cysteines is proposed. The consensus nucleotide sequence of this subdomain is also found in the MUC2 gene and in the MUC5B gene, two other mucin genes mapped to 11p15. The functional significance for secreted mucins of these cysteine-rich subdomains and the modular organization of mucin peptides are discussed.

Secretory differentiation and cell type identification of a human fetal bronchial epithelial cell line (HFBE).

A human fetal bronchial epithelial cell line (HFBE) grew in an undifferentiated pattern under conventional culture conditions. Despite a somewhat fibroblastic shape the cells maintained immunoreactivity to cytokeratin, carcinoembryonic antigen and epithelial membrane antigen. When grown on a collagen gel in a growth-hormone-supplemented medium, their spindle shape became more conspicuous. With an additional supplement of vitamin A (6 micrograms/ml), most of the cells underwent differentiation by producing many bright inclusion bodies which proved to be strongly positive with periodic acid-Schiff and weakly positive with alcian blue staining. Electron microscopy revealed a well-developed rough endoplasmic reticulum, an enlarged Golgi apparatus and many highly electron-dense secretory granules resembling those of Clara cells. Biochemical analysis demonstrated that HFBE cells cultured on collagen gel with vitamin A secreted hyaluronic acid and neutral glycoproteins containing mainly N-linked glycoproteins whose glycans were of a complex type. A monoclonal antibody (SEC-41) generated against the neutral glycoproteins detected a glycoprotein of approximately 52 kDa in the spent culture medium of differentiated HFBE cells. This antibody also reacted with the intracytoplasmic secretory granules in these cells. When tested on frozen sections of lung tissue, the immunohistochemical reactivity of the SEC-41 antibody was confined to Clara cells, some type II pneumocytes in the adult lung, and respiratory epithelial cells in the fetal lung. Moreover, this antibody could detect secretory glycoprotein in broncho-alveolar lavages from two patients. This paper clearly demonstrates that cells derived from human fetal bronchial epithelium can be cultivated in an undifferentiated precursor state and, under appropriate culture conditions, can be stimulated to undergo differentiation into a Clara cell type.

[Bronchopulmonary aminochlorthenoxycycline levels in experimental burns of the respiratory tree]. / Ricerche sul tropismo degli antibiotici a livello broncopolmonare nella ustione sperimentale dell'albero respiratorio.

A special technique was employed to obtain burns of the respiratory tree in 24 male rabbits, Increased bronchial secretion was observed during the burning. Aminochlorthenoxycycline values were higher in the secretion after 90' than in the controls, while blood and lung concentrations displayed a similar pattern. The usefulness of the drug in the prevention and treatment of infectious complications following burns in this area is stressed.