Tracheal Sections Research Articles

The effect of resveratrol in tracheal tissue of rats exposed to cigarette smoke

Objective: The aim of this study was to evaluate the effect of resveratrol on the tracheal tissue of rats exposed to cigarette smoke.Materials and methods: 40 adult Wistar albino rats were divided into four groups for an experiment of 6 weeks. Animals in group 1 were controls (n = 10). Rats in group 2 were exposed to cigarette smoke only, and rats in group 3 received daily intraperitoneal injections of resveratrol (10 mg/kg/d). Animals in group 4 were exposed to both cigarette smoke and intraperitoneal injections of resveratrol. Rats of all groups were sacrificed using cervical dislocation. The tracheas were removed and embedded in paraffin blocks. Sections of 4–5 μm thickness were prepared from the blocks. These sections were stained with hematoxylin and eosin, periodic acid–Schiff, and Alcian blue and viewed with a Leica DFC 280 light microscope.Results: Tracheal sections showed that, in group 2 (cigarette smoke group), there was desquamation of epithelial cells into the tracheal lumen, loss of cilia in the epithelial layer, an increase of goblet cells, activation of serous glands at the submucosa, and cell infiltration. In group 4 (cigarette smoke + resveratrol group), all these findings also existed but only a few sections were affected. It was observed that cigarette smoking caused morphological changes such as epithelial degeneration in the upper airway. These morphological changes were correlated with the amount of toxic substances in the cigarette smoke.Conclusion: We found that resveratrol had a preventive role in the histopathological changes caused by cigarette smoking in the rat trachea.

Adenosine A1 and A3 Receptor Agonists Inhibit Nonadrenergic, Noncholinergic Relaxations in the Guinea Pig Isolated Trachea

Background: Adenosine affects the tone and reactivity of airways by activating specific membrane receptors, named A₁, A_2a, A_2b and A₃. It affects cellular activities either directly by regulating membrane ion exchanges and polarization, or indirectly by modifying neurotransmitter release. Objectives: We assessed the effect of A₁ and A₃ receptor activation on electrically induced nonadrenergic, noncholinergic (NANC) relaxations in the guinea pig isolated trachea and the localization of A₁ and A₃ receptors in tracheal inhibitory neurons. Methods: NANC responses at 3 Hz were evaluat- ed in the presence of 2-chloro-N⁶-cyclopentyladenosine (CCPA), a selective A₁ agonist, and 2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA), a selective A₃ agonist, before and after the administration of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective A₁ antagonist, or 9-chloro-2-(2-furanyl)-5-((phenylacetyl)amino[1,2,4]triazolo[1,5-c])quinazoline (MRS 1220), a selective A₃ antagonist, respectively. For immunohistochemistry, tissues were exposed to antibodies to HuC/D, a general neuronal marker, neuronal nitric oxide synthase (nNOS), and A₁ or A₃ adenosine receptors and processed by indirect immunofluorescence. Results: CCPA (10 nM–3 μM) inhibited NANC relaxations. DPCPX (10 nM) failed to antagonize the effect of CCPA, but inhibited per se NANC relaxations (range 0.1–100 nM). CCPA (10 nM–10 μM) contracted unstimulated tracheal preparations, an effect antagonized by 10 nM DPCPX, with a pK_B value of 8.43. Cl-IB-MECA (10 nM–3 μM) inhibited NANC relaxations through a mechanism antagonized by MRS 1220 (100 nM). A₁- and A₃-positive neurons containing nNOS were detected in tracheal sections. Conclusions: Enogenous adenosine may induce airway hyperresponsiveness by inhibiting NANC relaxations via A₁ and A₃ receptors.

Potentiation of IL-19 expression in airway epithelia by IL-17A and IL-4/IL-13: Important implications in asthma

IL-17A and IL-19 are highly expressed in chronic inflammatory diseases, such as psoriasis and asthma. IL-19 plays a significant role in the enhancement of T(H)2 cytokine secretion in allergic diseases, but its cellular source in asthmatic patients remains unknown. Our aims were to determine whether the epithelium is a major source of airway mucosal IL-19 and to elucidate the mechanism of gene expression regulation. Immunofluorescent staining was used to determine IL-19 protein expression in tracheal tissue sections of various airway diseases. Well-differentiated primary human bronchial epithelial cultures and a corresponding cell line were used as in vitro models to study gene regulation. We found significantly higher IL-19 expression in airway epithelia of asthmatic patients than in epithelia of patients with other diseases. Using a cytokine panel, we demonstrated the upregulation of IL-19 expression in cultures by two T(H)2 cytokines, IL-4 and IL-13, in addition to the previously found T(H)17 cytokine IL-17A. Moreover, cotreatment of IL-17A and IL-4/IL-13 synergistically upregulated IL-19 expression. Using siRNA and chemical inhibitor approaches, we demonstrated a transcriptional regulation of IL-19 by nuclear factor kappaB and signal transducer and activator of transcription (STAT) 6. The addition of IL-13 to IL-17A stimulation triggers a shift from nuclear factor kappaB-dependent transcriptional regulation to one that is STAT6 based. Using chromatin immunoprecipitation assays, we demonstrated the presence of STAT6-binding elements in the IL-19 promoter region. We propose that an IL-17A- and IL-13-induced synergism in IL-19 stimulation in airway epithelia occurs through a STAT6-dependent pathway.

Hyaluronidase Expression and Activity Is Regulated by Pro-Inflammatory Cytokines in Human Airway Epithelial Cells

Hyaluronan (HA) is present at the apical surface of airway epithelium as a high-molecular-weight polymer. Since HA depolymerization initiates a cascade of events that results in kinin generation and growth factor processing, in the present work we used primary cultures of human bronchial epithelial (HBE) cells grown at the air-liquid interface (ALI) to assess hyaluronidase (Hyal) activity by HA zymography, gene expression by quantitative real-time PCR, and localization by confocal microscopy. Because TNF-alpha and IL-1beta induce Hyals in other cells, we tested their effects on Hyals expression and activity. We found that Hyal-like activity is present in the apical and basolateral secretions from HBE cells where Hyals 1, 2, and 3 are expressed, and that IL-1beta acts synergistically with TNF-alpha to increase gene expression and activity. Confocal microscopy showed that Hyals 1, 2, and 3 were localized intracellularly, while Hyal2 was also expressed at the apical pole associated with the plasma membrane, and in a soluble form on the apical secretions. Tissue sections from normal individuals and from individuals with asthma showed a Hyal distribution pattern similar to that observed on nontreated HBE cells or exposed to cytokines, respectively. In addition, increased expression and activity were observed in tracheal sections and in bronchoalveolar lavage (BAL) obtained from subjects with asthma when compared with normal lung donors and healthy volunteers. Our observations indicate that Hyal 1, 2, and 3 are expressed in airway epithelium and may operate in a coordinated fashion to depolymerize HA during inflammation associated with up-regulation of TNF-alpha and IL-1beta, such as allergen-induced asthmatic responses.

Calcium-mediated, purinergic stimulation and polarized localization of calcium-sensitive adenylyl cyclase isoforms in human airway epithelia

Purinergic stimulation of human airway epithelia results in a prolonged increase in ciliary beat frequency that depends on calcium-mediated cAMP production [Lieb, T., Wijkstrom Frei, C., Frohock, J.I., Bookman, R.J. and Salathe, M. (2002) Prolonged increase in ciliary beat frequency after short-term purinergic stimulation in human airway epithelial cells. J. Physiol. (Lond.) 538, 633–646]. Here, fully differentiated human airway epithelial cells in culture are shown to express calcium-stimulated transmembrane adenylyl cyclase (tmAC) isoforms (types 1, 3, and 8) by reverse transcription polymerase chain reaction. Immunohistochemistry of tracheal sections and fully differentiated airway epithelial cell cultures revealed polarized expression of these tmACs, with types 1 and 8 localized to the apical membrane and thus at the position required for ciliary regulation. Real-time, ciliated-cell specific cAMP production by tmACs upon apical, purinergic stimulation with UTP was confirmed using fluorescent energy resonance transfer between fluorescently tagged PKA subunits.

Role of carbon monoxide in electrically induced non-adrenergic, non-cholinergic relaxations in the guinea-pig isolated whole trachea.

Nitric oxide (NO) and vasoactive intestinal peptide (VIP) are considered transmitters of non-adrenergic, non-cholinergic (NANC) relaxations in guinea-pig trachea, whereas the role of carbon monoxide (CO) is unknown. This study was designed to assess the participation of CO, and to investigate the localization of haem oxygenase-2 (HO-2), the CO-producing enzyme, in tracheal neurons. NANC responses to electrical field stimulation (EFS) at 3 and 10 Hz were evaluated in epithelium-free whole tracheal segments as intraluminal pressure changes. Drugs used were: L-nitroarginine methyl ester (L-NAME, 100 microM) to inhibit NO synthase (NOS), alpha-chymotrypsin (2 U ml(-1)) to inactivate VIP, zinc protoporphyrin-IX (ZnPP-IX, 10 microM) to inhibit HO-2, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM), a soluble guanylyl cyclase inhibitor. For immunohistochemistry, tissues were exposed to antibodies to PGP 9.5, a general neuronal marker, HO-2 and NOS, and processed with an indirect immunofluorescence method. alpha-Chymotrypsin did not affect NANC relaxations. ODQ inhibited NANC responses by about 60%, a value similar to that obtained by combining L-NAME and ZnPP-IX. The combination of ODQ, L-NAME and ZnPP-IX reduced the responses by 90%. Subpopulations of HO-2 positive neurons containing NOS were detected in tracheal sections. In the guinea-pig trachea, NANC inhibitory responses at 3 and 10 Hz use NO and CO as main transmitters. Their participation is revealed following inhibition of NOS, HO-2 and soluble guanylyl cyclase. The involvement of CO as a relaxing transmitter paves the way for novel therapeutic approaches in the treatment of airway obstruction.

Regulated Hydrogen Peroxide Production by Duox in Human Airway Epithelial Cells

Hydrogen peroxide (H(2)O(2)) is found in exhaled breath and is produced by airway epithelia. In addition, H(2)O(2) is a necessary substrate for the airway lactoperoxidase (LPO) anti-infection system. To investigate the source of H(2)O(2) produced by airway epithelia, PCR was used to screen nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression in human airway epithelia redifferentiated at the air-liquid interface (ALI) and demonstrated the presence of Duox1 and 2. Western blots of culture extracts indicated strong expression of Duox, and immunohistochemistry of human tracheal sections localized the protein to the apical portion of epithelial cells. Apical H(2)O(2) production was stimulated by 100 microM ATP or 1 microM thapsigargin, but not 100 microM ADP. Diphenyleneiodonium, an NADPH oxidase inhibitor, and dimethylthiourea, a reactive oxygen species scavenger, both inhibited this stimulation. ATP did not stimulate the basolateral H(2)O(2) production by ALI cultures. ATP and thapsigargin increased intracellular Ca(2+) with kinetics similar to increasing H(2)O(2) production, and thus consistent with the expected Ca(2+) sensitivity of Duox. These data suggest that Duox is the major NADPH oxidase expressed in airway epithelia and therefore a contributor of H(2)O(2) production in the airway lumen. In addition, the data suggest that extracellular H(2)O(2) production may be regulated by stimuli that raise intracellular Ca(2+).

Spontaneous remission of edema and regranulation of goblet cells in rat tracheae after capsaicin-induced acute inflammation.

Previous studies have investigated the short-term effect of capsaicin on edema formation and goblet-cell secretion in the trachea. The present study sought to investigate the long-term effect of a high dose of capsaicin (90 micro g/ml/kg), administered intravenously, on changes in the formation of endothelial gaps among venular endothelial cells, mucosal tissue edema and the secretory activity of goblet cells, including the number and size of goblet cells, and the mucus score and secretory ratio of goblet-cell mucus secretion in the trachea of rats. The tracheal whole mounts with silver staining, those stained with chloroacetate esterase reagent and Alcian blue and tracheal tissue sections stained with Alcian blue and periodic acid-Schiff reagent were used for evaluation. Formation of endothelial gaps occurred a few min after administration of capsaicin, and gaps almost closed within 12 min after capsaicin injection. Five min after capsaicin, the leaky blood vessels were numerous and the subepithelial edema ratio (% of length of edema along the inner circumference of tracheal cross section) was found to be 57.8+/-3.0% ( n=6). The number of Alcian blue-positive goblet cells (1,090+/-220 per mm(2) of mucosal surface) was reduced to half the number of goblet cells in the vehicle-treated rats (2,200+/-230). The mucus score of goblet cell secretion was not changed. The secretory ratio was greatly increased. One day after capsaicin, the edema ratio remained large and the number of Alcian blue-positive goblet cells was also small. The mucus score was also not changed. The secretory ratio was still large. On day 3, the edema ratio remained large, but the number of Alcian blue-positive goblet cells was increased to the level of the controls. The mucus score and secretory ratio returned to the control level. On day 5, the edema ratio was greatly decreased, but it was still significantly larger than that of the controls. The mucus score and secretory ratio remained at the baseline level. Seven days after capsaicin, the edema ratio was similar to the controls. The number of goblet cells was even larger than controls. It is concluded that capsaicin-induced acute inflammation in the rat trachea involves formation of endothelial gaps, extensive plasma extravasation and edema formation, and depletion of goblet-cell secretory granules. Spontaneous gradual remission of edema was accompanied by regranulation of goblet cells with gradual mucogenesis for several days.

Measles virus preferentially transduces the basolateral surface of well-differentiated human airway epithelia.

Measles virus (MV) is typically spread by aerosol droplets and enters via the respiratory tract. The progression of MV infection has been widely studied; yet, the pathway for virus entry in polarized human airway epithelia has not been investigated. Herein we report the use of a replication-competent Edmonston vaccine strain of MV expressing enhanced green fluorescent protein (MV-eGFP) to infect primary cultures of well-differentiated human airway epithelia. Previous studies with polarized Caco-2 cells (intestine-derived human epithelia) and MDCK cells (kidney-derived canine epithelia) demonstrated that MV primarily infected and exited the apical surface. In striking contrast, our results indicate that MV preferentially transduces human airway cells from the basolateral surface; however, virus release remains in an apical direction. When MV-eGFP was applied apically or basolaterally to primary cultures of airway epithelia, discrete foci of eGFP expression appeared and grew; however, the cell layer integrity was maintained for the duration of the study (7 days). Interestingly, utilizing immunohistochemistry and confocal microscopy, we observed widespread expression of the receptor for the vaccine strain of MV (CD46) at greatest abundance on the apical surface of the differentiated human airway epithelia as well as in human tracheal tissue sections. These data suggest that the progression of MV infection through the respiratory epithelium may involve pathways other than direct binding and entry through the apical surface of airway epithelia.

Auxotonic Contractile Responses of Rat Tracheal and Bronchial Airway Smooth Muscle

The purposes of the present study were to directly compare: (1) the degree of trachealis muscle shortening and changes in tracheal dimensions and (2) ACh-mediated auxotonic contraction of trachea and intraparenchymal bronchi. The auxotonic contractile properties of tracheal and bronchial airway smooth muscle were assessed from 1–2 mm thick tracheal sections and 1 mm thick lung slices using videomicrometry in vitro at 37°C. Acetylcholine resulted in reductions in luminal area, perimeter, mean radius, length, and breadth (22.0, 10.0, 11.9, 10.7 and 12.0%, respectively). Trachealis muscle shortening reached a maximum of 39.8±4.3%. The K+channel blocker 4-aminopyridine significantly augmented the ACh-mediated reductions in tracheal luminal dimensions. In response to ACh (10−3m), reductions in bronchial dimensions were significantly greater than those of the trachea for luminal area, perimeter and mean radius (44.6 vs. 18.6, 32.0 vs. 8.0 and 28.9 vs. 9.9%, respectively). These data indicate that auxotonic contractile responses of rat tracheal smooth muscle differ from those previously reported in the dog and guinea pig, that ACh-mediated auxotonic contraction of tracheal smooth muscle is augmented by 4-aminopyridine, and that proportionate reductions in luminal dimensions in response to ACh are considerably greater for bronchial than tracheal airways.

Proinflammatory characteristics of a nonpeptide bradykinin mimic, FR190997, in vivo.

1. Proinflammatory potency of the nonpeptide bradykinin (BK) B(2) receptor agonist FR190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl)cinnamidoacetyl]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoline) was investigated. 2. Intradermal injection of FR190997 (0.03 - 3 nmol site(-1)) into dorsal skin of rats increased vascular permeability in a dose-dependent manner. The effect was less than that of BK, but it was long-acting and was inhibited by treatment with FR173657 (3 mg kg(-1), p.o.). Captopril (10 mg kg(-1), i.p.) did not enhance the plasma extravasation by FR190997 (0.3 nmol site(-1)) in the presence of soybean trypsin inhibitor (SBTI, 30 microg site(-1)). 3. Subcutaneous injection of FR190997 (3 nmol site(-1)) into the hindpaw of mice markedly induced paw swelling. The oedema lasted up to 3 h after the injection. Administration of indomethacin or NS-398 (10 mg kg(-1), i.p.) significantly reduced it at 3 h after the injection. 4. Simultaneous i.p. injection of prostaglandin (PG) E(2) (1 nmol site(-1)) or beraprost sodium (0.5 nmol site(-1)) with FR190997 (5 nmol site(-1)) greatly enhanced frequency of writhing reactions in mice. 5. FR190997 (0.3 - 30 nmol kg(-1), i.v.) showed less increase in airway opening pressure (Pao) in the guinea-pig after i.v. injection. Furthermore, FR190997 (0.03 - 30 nmol) resulted in a very weak contraction of tracheal ring strips and lung parenchymal sections in vitro. 6. In mice sponge implants, topical application of FR190997 increased angiogenesis and granulation with enhanced expressions of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) mRNAs. 7. These results indicate that FR190997 has proinflammatory long-lasting characteristics and it might be 'a stable tool' for studying the role of BK B(2) receptor in vivo.

Isolation and characterization of a capsule-deficient mutant of Actinobacillus pleuropneumoniae serotype 1

The capsular polysaccharides (CPS) play a major role in pathogenicity of Actinobacillus pleuroÍpneumoniae, the causative agent of porcine pleuropneumonia. The purpose of the present study was to isolate a mutant in CPS biosynthesis by using a mini-Tn 10 transposon mutagenesis system and evaluate its adherence to host cells. One mutant apparently did not possess CPS as it did not react with a monoclonal antibody against A. pleuropneumoniae serotype 1 capsular antigen. Absence of capsule was confirmed by flow cytometry and also by transmission electron microscopy after polycationic ferritin labelling. The site of insertion of the mini-Tn 10 was determined and found to be in thecpxC gene. Its gene product, CpxC, is a protein involved in polysaccharide transport across the cytoplasmic membrane during CPS biosynthesis. Use of piglet tracheal frozen sections indicated that the CPS mutant adhered significantly (P=0.0001) more than the parent strain. The non-capsular mutant was less virulent in pigs compared to the parent strain and showed no mortality in experimentally infected pigs. The CPS mutant was however resistant to pig serum. This CPS mutant is the first A. pleuropneumoniae mutant in a CPS transport gene. It is also the first time that adherence of a CPS mutant of A. pleuropneumoniae is evaluated. Our observations indicate that capsular polysaccharides of A. pleuropneumoniae serotype 1 are not involved in adherence to piglet tracheal frozen sections but rather mask, at least in part, the adhesive functions.

Inhibition of adherence of Actinobacillus pleuropneumoniae to porcine respiratory tract cells by monoclonal antibodies directed against LPS and partial characterization of the LPS receptors.

Actinobacillus pleuropneumoniae is the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia. We have previously identified the lipopolysaccharides (LPS) as the major adhesin of A. pleuropneumoniae involved in adherence to porcine respiratory tract cells. In the present study, adherence of A. pleuropneumoniae to porcine tracheal frozen sections was inhibited by homologous monovalent Fab fragments produced from monoclonal antibodies 5.1 G8F10 and 102-G02 directed, respectively, against the A. pleuropneumoniae serotype 1 or serotype 2 O-antigens. These results confirm the important role played by LPS in adherence of A. pleuropneumoniae and suggest that these adhesins might represent good vaccine candidates. We also investigated the presence of A. pleuropneumoniae receptors in tracheal cell preparations from piglets of four different breeds. Using Far-Western binding assays, we identified proteins recognized by whole cells of A. pleuropneumoniae reference strains for serotype 1 and 2, and local isolates belonging to the same serotypes, and also recognized by extracted LPS from both reference strains. We confirmed the proteinaceous nature of these LPS-binding molecules by their staining with Coomassie brilliant blue, sensitivity to proteinase K digestion, resistance to sodium m-periodate oxidation, and their inability to stain with glycoprotein-specific reagents. Four low-molecular-mass bands (14-17 kDa) seemed to correspond to histones. We also identified proteins at Mr 38,500 that could represent putative receptors for A. pleuropneumoniae LPS in swine respiratory tract cells.

Isolation and characterization of mini-Tn10 lipopolysaccharide mutants of Actinobacillus pleuropneumoniae serotype 1.

Lipopolysaccharide (LPS) has previously been identified as the major adhesin of Actinobacillus pleuropneumoniae involved in adherence to porcine respiratory tract cells. The purpose of the present study was to isolate and characterize mutants in LPS biosynthesis by using a mini-Tn10 transposon mutagenesis system. Seven mutants appeared to possess a rough LPS (among which two had similar Southern blot profiles) while one mutant (#5.1) expressed the high-molecular-mass LPS, but as visualized by Tricine SDS-PAGE, showed an additional band in the core-lipid A region. The LPS mutants showed sensitivity to pig serum to various degrees, while the parent strain was serum-resistant. Use of piglet frozen tracheal sections indicated that, surprisingly, the rough LPS mutants adhered similarly or in greater numbers than the parent strain. However, the LPS mutant #5.1 adhered significantly less than the parent strain and was also less virulent in pigs. The gene affected by mini-Tn10 in LPS mutant #5.1 is galU, the structural gene for UTP-alpha-D-glucose-1-phosphate uridylyltransferase, involved in LPS core biosynthesis. Complementation analysis confirmed that the phenotypic characteristics of LPS mutant #5.1 are the result of the inactivation of the galU gene. Our data suggest that although the presence of O-antigen does not seem to be essential, an intact core-lipid A region might be required for adherence of A. pleuropneumoniae to porcine respiratory tract cells. To the best of our knowledge, these mutants represent the first isogenic mutants of A. pleuropneumoniae defective in LPS biosynthetic genes.

Matrix metalloproteinase gelatinases in sulfur mustard-induced acute airway injury in guinea pigs

Respiratory tract lesions induced by sulfur mustard (SM), a chemical warfare agent, are characterized by epithelial damage associated with inflammatory cell infiltration. To test the potential role of matrix metalloproteinase gelatinases in these lesions, we evaluated gelatinase activity, albumin content, and total cell count in bronchoalveolar lavage fluid of guinea pigs 24 h after an intratracheal injection of 0.2 mg/kg of SM. The bronchial lavage and alveolar lavage fluids were analyzed separately. The increase in inflammatory cell content of the bronchial lavage fluid, mainly macrophages, observed in SM-intoxicated guinea pigs was accompanied by an increase in albumin and in 92-kDa gelatinase activity. There was a significant correlation between albumin content and 92-kDa gelatinase activity (r = 0.67) and between 92-kDa gelatinase and the number of macrophages. Immunohistochemistry performed on tracheal sections showed the presence of 92-kDa gelatinase at the site of intraepithelial cleavages. Zymography analysis of culture medium conditioned by guinea pig tracheal epithelial cells demonstrated that these cells produced in vitro 92-kDa gelatinase on stimulation. Culture of human bronchial epithelial cells obtained by the explant technique showed a marked increase in 92-kDa gelatinase after exposure to 5 x 10(-5) M SM that reinforced the relevance of our animal results to human exposure to SM. These results suggest that in SM respiratory intoxication, 92-kDa gelatinase of both inflammatory and epithelial cell origins could be involved in epithelial cell detachment.

Antigen-induced elevation of immunoreactive endothelin-1 (ET-1) levels in ovalbumin-sensitized guinea pig airway tissue

Changes in the immunoreactive ET-1 levels during the anaphylactic reaction of airway tissue from ovalbumin-sensitized guinea pigs were investigated. ET-1-immunoreactivity (ET-IR) was detected in the epithelial and smooth muscle layers of tracheal sections from normal guinea pigs and it was enhanced slightly by phosphoramidon (1 μM) treatment. The ET-IR level of the epithelial layer of ovalbumin-treated tissue from actively sensitized animals was slightly higher than that from normal animals, but it was enhanced markedly by phosphoramidon (1 μM) treatment. Furthermore, the mean ET-IR level of homogenates of antigen-treated tracheal tissues from sensitized guinea pigs (22.8±1.55 fmol mg −1 protein, n=5) was significantly higher than the corresponding normal level (12.3±1.21 fmol mg −1 protein, n=5). These results suggest that increased epithelial airway ET-1 levels contribute to the anaphylactic reaction of guinea pig airways.

Inclusion Body Tracheitis Associated with Avian Adenovirus in Turkeys

Nine turkey flocks with basophilic intranuclear inclusion bodies, suggestive of adenovirus, within the epithelial cells of the tracheal mucosa were studied. Respiratory signs and increased mortality occurred in turkeys between 6 and 10 wk of age from nine commercial turkey meat flocks in central California. Necropsy findings included tracheitis and occasional mild sinusitis. Histopathology of the tracheas revealed epithelial deciliation, squamous metaplasia, large basophilic intranuclear inclusion bodies within epithelial cells, and lymphoplasmatic inflammation in the mucosa. Electron microscopy of the mucosa revealed hexagonal viral particles, approximately 73 nm in diameter, consistent with adenovirus within the nuclei of epithelial cells. All tracheal sections were negative for adenovirus group II by specific immunoperoxidase staining. Adenovirus group I was isolated from the trachea. In addition, Bordetella avium, Ornithobacterium rhinotracheale, and Klebsiella pneumoniae were isolated from the tracheas of three, three, and two flocks, respectively. Statistically greater total mortality and a smaller percentage of marketed turkeys were observed in the submitted flocks than in randomly selected flocks. No significant difference was observed between the two turkey groups in market weight, feed conversion, or percentage of grade "A" turkeys.

Elevation of Endogenous Nucleophiles in Rat Lung by Cysteine and Glutathione Esters In Vitro

In this study, we have compared the uptake of l-cysteine ( l-CySH), d-cysteine ( d-CySH), l-cysteine isopropyl ester ( l-CIPE) and d-cysteine isopropyl ester ( d-CIPE) in rat lung slices and tracheal sections and determined the effectiveness of glutathione (GSH), GSH isopropyl monoester, GSH isopropyl diester, γ-glutamylcysteine (γ-glu-cys) isopropyl monoester and γ-glu-cys isopropyl diester to elevate and prolong intracellular GSH concentrations in rat lung slices. Lung slices were incubated with 1.0 mM of thiol and the concentrations determined intracellularly and extracellularly with time. Slices incubated with GSH, GSH isopropyl diester and γ-glu-cys isopropyl diester had cellular GSH concentrations increased by up to 60%, 95% and 58%, respectively, whereas GSH isopropyl monoester and γ-glu-cys isopropyl monoester did not increase the intracellular GSH concentration. Extracellularly, the GSH concentration had decreased by 15%, GSH isopropyl diester by 27%, γ-glu-cys isopropyl diester by 66% and both isopropyl monoesters by over 90% at 120 min. Lung slices and tracheal sections incubated with l- or d-CySH at 37° had increased cellular concentrations of l- and d-CySH which ranged between 0.88–1.25 nmol mg −1 and 1.35–2.25 nmol mg −1, respectively. Reducing the incubation temperature to 4° had little effect on the accumulation of d-CySH; however, l-CySH concentrations increased progressively in the trachea and lung to reach 2.73 and 2.63 nmol mg −1 at 90 min, respectively. Lung slices incubated with l- or d-CIPE had increased l- or d-CySH concentrations up to a max of 13.7 and 11.1 nmol mg −1 and tracheal sections up to a max of 5.56 and 11.09 nmol mg −1. In the lung slice medium, l- and d-CIPE levels had decreased by 75.2% and 74.0% at 90 min, respectively, and from the tracheal section medium, l- and d-CIPE concentrations had decreased by 66.7% and 32.7%, respectively. Preincubation of lung slices and tracheal sections with the carboxylesterase inhibitor, bis ( p-nitrophenyl) phosphate (BNPP), almost completely prevented the disappearance of l- and d-CIPE extracellularly and greatly reduced the appearance of cellular l- and d-CySH. GSH, GSH isopropyl diester and γ-glu-cys isopropyl diester elevated and prolonged GSH concentrations in rat lung slices, but GSH isopropyl monoester and γ-glu-cys isopropyl monoester did not increase GSH levels. The uptake of l-CySH, but not d-CySH, is temperature sensitive in rat lung slices and tracheal sections and carboxylesterases appear to have a major influence on the uptake and metabolism of l- and d-CIPE by rat lung slices and tracheal sections.

Effect of Inhaled Cyclosporin on the Rat Airway: Histologic and Bronchoalveolar Lavage Assessment

Airway inflammation plays a pivotal role in asthma. Over the last 10 years, evidence has accumulated for the potential role of lymphocytes in airway inflammation. Since cyclosporin A (Cyc-A) can profoundly influence lymphocyte activation, it is appropriate to consider this drug as a novel antiasthmatic. The effect of inhalation of low doses of Cyc-A on airway inflammation remains unclear. The purpose of this study was to investigate the bronchoalveolar lavage (BAL), peripheral blood cell profiles, and lung biopsy specimens in Cyc-A-pretreated rats. Twenty-nine rats (8 controls, 10 ovalbumin sensitized, and 11 Cyc-A inhaling and ovalbumin sensitized) were included in the study. A commercial intravenous Cyc-A solution was given as a single dose of 20 mg/kg 1 h prior to inhalation of ovalbumin via nebulizer. The total number of BAL cells significantly increased in rats inhaling Cyc-A when compared with ovalbumin-sensitized rats (2.37 ± 2.34 × 10⁶/ml and 1.01 ± 0.49 × 10⁶/ml respectively, p < 0.05). There was a significant increase in the percentage of lymphocytes (14.5 ± 8.5 versus 27.4 ± 7.4%, p < 0.03), a nonsignificant increase in the percentage of eosinophils (0.8 ± 1.0 versus 3.0 ± 4.6%), and a significant decrease in the percentage of polymorphonuclear leukocytes (9.4 ± 6.9 versus 3.4 ± 3.8%, p < 0.01) and macrophages (75.4 ± 5.1 versus 50.2 ± 11.8%, p < 0.02) in BAL in the ovalbumin-sensitized group as compared with controls. Differential cell counts revealed a higher percentage of neutrophils and macrophages in the BAL of Cyc-A-pretreated rats than in that of the ovalbumin-sensitized group (26.3 ± 26.8 versus 3.4 ± 3.8%, p < 0.01 and 66.1 ± 7.7 versus 50.2 ± 11.8%, p < 0.02). There was a nonsignificant decrease of lymphocytes and eosinophils in the Cyc-A-pretreated group when compared with the ovalbumin-sensitized group (27.4 ± 7.4 versus 21.1 ± 12.4 and 3.0 ± 4.6% versus 2.4 ± 2.6%). The peripheral blood total white blood cell count decreased in the ovalbumin-sensitized and Cyc-A-pretreated groups as compared with the control group (2,520 ± 1,098/mm³, 3,591 ± 2,251/mm³, and 5,975 ± 2,787/mm³, respectively, p < 0.01). In addition, peripheral eosinophilia was detected in the Cyc-A-pretreated group when compared with controls and the ovalbumin-sensitized group (6.9 ± 4.7, 2.4 ± 1.1, and 2.6 ± 2.4%, respectively, p < 0.01). Light-microscopic examination of the airways revealed prominent eosinophilia in tracheal, bronchial, and bronchiolar sections in the ovalbumin-sensitized group: counts were 1.8 ± 2.3/HPF, 10.3 ± 11.4/HPF, 63.3 ± 45.0/HPF, respectively. Cyc-A resulted in a decrease of the eosinophil counts/HPF to 0/HPF in trachea (p < 0.05), to 4.3 ± 9.4/HPF in bronchi (p < 0.02), to 19.4 ± 38.4 in bronchioles (p < 0.004).In conclusion, the present study supports the theory that locally administered inhaled low-dose Cyc-A is effective on inflammatory cells of sensitized airways and peripheral cells. It may therefore be useful in elucidating the inflammatory mechanisms involved in asthma.

Isolation and characterization of LPS mutants of Actinobacillus pleuropneumoniae serotype 1.

The major adhesin of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, has been previously identified as lipopolysaccharide (LPS). The purpose of the present study was to isolate and characterize A. pleuropneumoniae LPS mutants. Screening of LPS mutants was performed with colony dot and sensitivity to novobiocin. One mutant obtained by colony dot (F19) and one mutant selected for its increased sensitivity to novobiocin (33.1) did not react with a monoclonal antibody against A. pleuropneumoniae serotype 1 O-antigen compared with the parent strain. Mutants F19 and 33.1 did not express high-molecular-mass LPS bands as determined in silver-stained SDS-PAGE gels. The core-lipid A region of mutant 33.1 and of the parent strain had similar relative mobilities and reacted with serum from a pig experimentally infected with the serotype 1 reference strain of A. pleuropneumoniae, while the same region in mutant F19 showed faster migration and did not react with this serum. Use of piglet tracheal frozen sections indicated that mutant F19 was able to adhere to piglet trachea as well as the parent strain, while mutant 33.1 adhered [half as much as] the parent strain. Finally, both LPS mutants were markedly less virulent in mice than the parent strain. Taken together, our observations support the idea that LPS is an important virulence factor of A. pleuropneumoniae.