Respiratory Tract Epithelial Cells Research Articles

All-trans retinoic acid mediates DUOX2 expression and function in respiratory tract epithelium

DUOX1 and DUOX2 are members of the NADPH oxidase family that are specifically regulated to produce hydrogen peroxide in epithelia of the thyroid, gastrointestinal tract, and respiratory tract. The determinants of DUOX1 or DUOX2 expression in various tissues have not been established. Using respiratory tract epithelial cells as a model, we investigated changes in DUOX mRNA and protein expression during the first 10 days of differentiation. By comparing a respiratory tract cell line, HBE1, with primary tracheobronchial epithelial (TBE) cells, we determined that DUOX2 was significantly expressed only in cell conditions that included all-trans retinoic acid (ATRA). In HBE1 cells, DUOX2 mRNA increased 6-fold after ATRA treatment. Similarly, ATRA induced a 19-fold increase in DUOX2 mRNA expression in primary TBE cells with parallel increases in DUOX protein and DUOX-mediated H(2)O(2) production as well. In addition, DUOX2 induction by rhinovirus required the presence of ATRA. ATRA had no effect on DUOX1 expression for all the conditions studied. Our data indicate that for respiratory epithelial cells, ATRA is important in the regulation of DUOX2 expression, function, and rhinovirus-mediated DUOX2 inducibility.

Topical curcumin can inhibit deleterious effects of upper respiratory tract bacteria on human oropharyngeal cells in vitro: potential role for patients with cancer therapy induced mucositis?

Curcumin exerts its anti-inflammatory activity via inhibition of nuclear factor κB. Oropharyngeal epithelia and residing bacteria closely interact in inflammation and infection. This in vitro model investigated the effects of curcumin on bacterial survival, adherence to, and invasion of upper respiratory tract epithelia, and studied its anti-inflammatory effect. We aimed to establish a model, which could offer insights into the host-pathogen interaction in cancer therapy induced mucositis. Moraxella catarrhalis (Mcat) and the oropharyngeal epithelial cell line Detroit 562 were used. Time-kill curves assessed the inhibition of bacterial growth and adherence assays and gentamicin protection assays determined the effect of curcumin-preincubated cells on bacterial adherence and invasion. Curcumin-mediated inhibition of pro-inflammatory activation by Mcat was determined via interleukin-8 concentrations in the supernatants. The synergistic role of secretory IgA (sIgA) on adherence was investigated. Curcumin was bactericidal at concentrations >50µM. Preincubation of Detroit cells for 60min demonstrated that concentrations >100µM inhibited bacterial adherence. Together with sIgA, curcumin inhibited adherence at concentrations ≥50µM. Both 100 and 200µM curcumin significantly inhibited Mcat cell invasion. Finally, curcumin inhibited Mcat-induced pro-inflammatory activation by strongly suppressing IL-8 release. At a concentration of 200µM, 10min of curcumin exposure inhibited IL-8 release significantly, and complete suppression required a pre-exposure time of ≥45min. Curcumin, in clinically relevant concentrations for topical use, displayed strong antibacterial effect against a facultative upper respiratory tract pathogen by inhibiting bacterial growth, adherence, invasion, and pro-inflammatory activation of upper respiratory tract epithelial cells in vitro.

Outer membrane porin M35 of Moraxella catarrhalis mediates susceptibility to aminopenicillins

BackgroundThe outer membrane protein M35 is a conserved porin of type 1 strains of the respiratory pathogen Moraxella catarrhalis. It was previously shown that M35 is involved in the uptake of essential nutrients required for bacterial growth and for nasal colonization in mice. The aim of this study was (i) to characterize the potential roles of M35 in the host-pathogen interactions considering the known multifunctionality of porins and (ii) to characterize the degree of conservation in the phylogenetic older subpopulation (type 2) of M. catarrhalis.ResultsIsogenic m35 mutants of the type 1 strains O35E, 300 and 415 were tested for their antimicrobial susceptibility against 15 different agents. Differences in the MIC (Minimum Inhibitory Concentration) between wild-type and mutant strains were found for eight antibiotics. For ampicillin and amoxicillin, we observed a statistically significant 2.5 to 2.9-fold MIC increase (p < 0.03) in the m35 mutants. Immunoblot analysis demonstrated that human saliva contains anti-M35 IgA. Wild-type strains and their respective m35 mutants were indistinguishable with respect to the phenotypes of autoagglutination, serum resistance, iron acquisition from human lactoferrin, adherence to and invasion of respiratory tract epithelial cells, and proinflammatory stimulation of human monocytes. DNA sequencing of m35 from the phylogenetic subpopulation type 2 strain 287 revealed 94.2% and 92.8% identity on the DNA and amino acid levels, respectively, in comparison with type 1 strains.ConclusionThe increase in MIC for ampicillin and amoxicillin, respectively, in the M35-deficient mutants indicates that this porin affects the outer membrane permeability for aminopenicillins in a clinically relevant manner. The presence of IgA antibodies in healthy human donors indicates that M35 is expressed in vivo and recognized as a mucosal antigen by the human host. However, immunoblot analysis of human saliva suggests the possibility of antigenic variation of immunoreactive epitopes, which warrants further analysis before M35 can be considered a potential vaccine candidate.

Unimpaired Lysosomal Acidification in Respiratory Epithelial Cells in Cystic Fibrosis

The mechanisms remain uncertain by which mutations in CFTR cause lung disease in cystic fibrosis (CF). Teichgräber et al. recently reported increased ceramide in CF lungs, which was proposed to result from defective lysosomal acidification in airway epithelial cells and consequent impairment of pH-dependent ceramide-metabolizing enzymes (Teichgräber, V., Ulrich, M., Endlich, N., Reithmüller, J., Wilker, B., Conceição Ce Olivereira-Munding, C., van Heeckeren, A. M., Barr, M. L., von Kürthy, G., Schmid, K. W., Weller, M., Tümmler, B., Lang, F., Grassme, H., Döring, G., and Gulbins, E. (2008) Nat. Med. 14, 382-391). Here, we measured lysosomal pH in several CFTR-expressing and -deficient cell lines, freshly isolated airway epithelial cells from non-CF and CF mice and humans, and well-differentiated primary cultures of human non-CF and CF airway epithelial cells. Lysosomal pH was measured by ratio imaging using a fluorescent pH indicator consisting of 40-kDa dextran conjugated to Oregon Green 488 and tetramethylrhodamine. In all cell types, lysosomal pH was approximately 4.5, unaffected by the thiazolidinone CFTR inhibitor CFTR(inh)-172, and increased to approximately 6.5 following bafilomycin inhibition of the vacuolar proton pump. Lysosomal pH did not differ significantly in airway epithelial cells from non-CF versus CF humans or mice. Our results provide direct evidence against the conclusions of Teichgräber et al. that lysosomal acidification is CFTR-dependent, impaired in CF, or responsible for ceramide accumulation. As such, alternative mechanisms are needed to explain increased ceramide in CF airways. Non-CFTR mechanisms, such as ClC-type chloride channels, are likely involved in maintaining electroneutrality during organellar acidification.

Nitroxide radical TEMPO reduces ozone-induced chemokine IL-8 production in lung epithelial cells

Exposure to high levels of ozone (O 3) damages respiratory tract epithelial cells. This research evaluated the ability of TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), a stable nitroxide free radical, to decrease O 3-mediated injury to a respiratory tract-derived cell line (A549 cells) by monitoring in this cell system the interleukine-8 (IL-8) production. TEMPO reduced O 3-induced IL-8 production in A549 cells, as evidenced by PCR analysis, Western blot and ELISA assays. This behaviour is explainable on the basis of the reactivity between TEMPO with O 3 and/or O 3-derived free radicals in biological systems. The study provides evidence that TEMPO reacts with O 3 and/or its cytotoxic products and may provide protections against O 3-induced biotoxicities.

Streptococcus pneumoniae early response genes to human lung epithelial cells

BackgroundStreptococcus pneumoniae infection starts from colonization of the host respiratory tract where interaction with host respiratory tract epithelial cells occurs. To investigate pneumococcal genes that are involved in the early stage of interaction with host epithelial cells, transcriptional responses of an encapsulated pathogenic pneumococcal strain TIGR4 upon exposure to human lung epithelial cells A549 for 0.5 h and 1 h time periods were investigated by using TIGR (JCVI) microarray technology. Gene expression changes were validated by quantitative real-time PCR (qRT-PCR) analysis.FindingsWe observed different transcriptional profiles at two incubation time periods in which most gene expressions were down-regulated at 0.5 h but up-regulated at 1 h. Many genes associated with ribonucleotide biosynthesis were down-regulated at both time points, whereas the genes associated with cell envelope, energy metabolism, transport and protein synthesis were mostly up-regulated at 1 h. Furthermore, these profiles were compared to the transcriptomes of a TIGR4-derived strain in response to human macrophages for the same time periods. We found one set of genes that exhibited similar expression changes upon exposure to both types of host cells, including cell envelope-associated bgaA (SP0648) and nanA (SP1693), and uncharacterized gene clusters such as SP1677–SP1680 and SP1688–SP1690.ConclusionThese data indicate that at the early stage of interaction with host epithelial cells, a complex gene regulation and expression change occur in bacteria. Some of them might play an essential role during pathogen-host interactions and for the establishment of infection.

Recent Anti-Influenza Strategies in Multivalent Sialyloligosaccharides and Sialylmimetics Approaches

Sialic acid-containing oligosaccharides expressed on the respiratory tract epithelial cell surface are involved in influenza virus infection in both virus attaching and detaching processes. Therefore, inhibition of sialic acid-binding processes provides rational anti-influenza strategies. Previous exploring efforts using monosaccharide sialic acid-bearing macromolecules provided proof of concept for multivalent hemagglutinin inhibition. However, the monosaccharide sialic acid cannot account for the molecular determinant of virus receptor-binding specificity in the context of the whole sialyloligosaccharide receptor. On the other hand, neuraminidase inhibition efforts using sialylmimetics have resulted into two antiinfluenza drugs, zanamivir and oseltamivir, which have been shown to reduce both the severity and duration of influenza illness. Nevertheless, the usage of monomeric sialylmimetics requires relatively large amounts of expensive compounds, which may also induce virus resistance and side effect. Therefore, it is critical to develop new antiinfluenza drugs and improve the current antiinfluenza drugs. This review highlights recent explorations of multivalent sialyloligosaccharides-based influenza virus adhesion inhibition strategy and multivalent sialylmimetics-based influenza virus detachment inhibition strategy for these efforts.

Effect of cigarette smoke extract on lipopolysaccha-ride-activated mitogen-activated protein kinase signal transduction pathway in cultured cells

Background Lipopolysaccharide (LPS) forms outer membrane of the wall of Gram-negative cells. LPS can directly cause damage to epithelia of respiratory tract and is the major factor responsible for the chronic inflammation of respiratory passage. The mitogen-activated protein kinase (MAPK) signal transduction pathway of the airway epithelia is intimately associated with the action of LPS. The chronic inflammation of respiratory tract and smoking are interrelated and entwined in the development and progression of chronic lung diseases. This study was designed to examine the effects of cigarette smoke extract (CSE) and LPS on MAPK signal transduction pathway in order to further understand the roles CSE and LPS play in chronic lung inflammation. Methods Cultured primary human epithelial cells of airway were divided into four groups according to the stimulants used: blank control group, LPS-stimulation group, CSE-stimulation group and CSE plus LPS group. Western blotting was employed for the detection of phosphorylation level of extracellular-signal-regulated-kinase (ERK1/2), p38 MAPK and c-Jun N-terminal kinase (JNK). The expression of cytokines of MAPK transduction pathway (granulocyte-macrophage colony stimulating factor (GM-CSF) and mRNA of IL-8) in the primary epithelial cells of respiratory tract was also determined. Results Western blotting revealed that the phosphorylation levels of ERK1/2, p38 MAPK and JNK were low and 2 hours after the LPS stimulation, the phosphorylation of ERK1/2, p38 MAPK and JNK were all increased. There was a significant difference in the phosphorylation between the LPS-stimulation group and blank control group (P<0.05); no significant difference was found between CSE-stimulation group and blank control group (P>0.05); there was a significant difference between CSE + LPS group and blank control group and between CSE + LPS group and LPS group (P<0.05). The phosphorylation of CSE-LPS group was higher than that of blank control group but lower than that of LPS group. In blank control group, the expression of IL-8 and GM-CSF mRNA was low in the epithelial cells of airway and the release of IL-8 and GM-CSF was also at a low level. One hour after LPS stimulation, the level of IL-8 mRNA increased (P<0.05) and reached a peak after 2 hours. On the other hand, GM-CSF mRNA level increased 2 hours after the stimulation (P<0.05) and reached the highest level 4 hours after the stimulation. Two hours after LPS stimulation, IL-8 and GM-CSF protein level began to rise (P<0.05), and the level was the highest 8 hours after the stimulation (P<0.01). Stimulation with CSE alone had no effect on the release of IL-8 and GM-CSF and expression of IL-8 mRNA (P>0.05), but pre-treatment with CSE could delay the LPS-induced release of IL-8 and GM-CSF and the expression of IL-8 mRNA and its peak was lower. Conclusions LPS stimulation can significantly increase the phosphorylation of ERK1/2, p38 MAPK and JNK in the epithelial cells of airway and activate the MAPK transduction pathway, thereby can activate the downstream signal transduction pathway, and can ultimately result in the release of cytokines by the epithelial cells of airway. CSE can partially abolish the LPS-induced activation of MAPK signal transduction pathway and the expression of cytokines of the pathway, which might contribute to the development and progression of the inflammatory reactions in COPD patients. Chin Med J 2007;120(12):1075–1081

Reduced Ability of Penicillin to Eradicate Ingested Group A Streptococci from Epithelial Cells: Clinical and Pathogenetic Implications

Group A streptococci (Streptococcus pyogenes; GAS) invades human epithelial cell lines. Failure of penicillin to eradicate GAS from the throats of patients, especially those who are GAS "carriers," has been increasingly reported. However, there has been no comprehensive evaluation of how effectively antibiotics that are used to treat GAS enter upper respiratory tract epithelial cells and kill internalized GAS. We examined the viability of ingested, intracellular GAS after epithelial cell exposure to antibiotics commonly recommended for therapy of GAS infections. A human laryngeal epithelial cell line (HEp-2) was used. Three techniques were used to study antibiotic (penicillin V, erythromycin, azithromycin, cephalothin, and clindamycin) killing of ingested GAS: examination by electron microscopy of ultrathin sections of ingested GAS, qualitative determination of intra-epithelial cell antibiotic, and special stain evaluation of intracellular GAS viability after epithelial cell exposure to antibiotics. GAS survived intracellularly despite exposure of the GAS-containing epithelial cells to penicillin. In contrast, there was killing of ingested GAS after exposure of epithelial cells to either erythromycin or azithromycin. Electron microscopy confirmed a lack of intracellular GAS fragmentation (cell death) after exposure of epithelial cells to penicillin in contrast to obvious GAS fragmentation after epithelial cell exposure to erythromycin or azithromycin. Cephalothin, a cephalosporin, and clindamycin were more effective in killing ingested GAS than was penicillin, but they were less effective than erythromycin or azithromycin. These observations strongly suggest that if the GAS upper respiratory tract carrier state results from intra-epithelial cell GAS survival, the failure of penicillin to kill ingested GAS may be related to a lack of effective penicillin entry into epithelial cells. These unique observations may have clinical implications for understanding GAS respiratory tract carriers and managing GAS infections.

Effects of Psa and F1 on the Adhesive and Invasive Interactions ofYersinia pestiswith Human Respiratory Tract Epithelial Cells

Yersinia pestis, the causative agent of plague, expresses the Psa fimbriae (pH 6 antigen) in vitro and in vivo. To evaluate the potential virulence properties of Psa for pneumonic plague, an Escherichia coli strain expressing Psa was engineered and shown to adhere to three types of human respiratory tract epithelial cells. Psa binding specificity was confirmed with Psa-coated polystyrene beads and by inhibition assays. Individual Y. pestis cells were found to be able to express the capsular antigen fraction 1 (F1) concomitantly with Psa on their surface when analyzed by flow cytometry. To better evaluate the separate effects of F1 and Psa on the adhesive and invasive properties of Y. pestis, isogenic Deltacaf (F1 genes), Deltapsa, and Deltacaf Deltapsa mutants were constructed and studied with the three respiratory tract epithelial cells. The Deltapsa mutant bound significantly less to all three epithelial cells compared to the parental wild-type strain and the Deltacaf and Deltacaf Deltapsa mutants, indicating that Psa acts as an adhesin for respiratory tract epithelial cells. An antiadhesive effect of F1 was clearly detectable only in the absence of Psa, underlining the dominance of the Psa+ phenotype. Both F1 and Psa inhibited the intracellular uptake of Y. pestis. Thus, F1 inhibits bacterial uptake by inhibiting bacterial adhesion to epithelial cells, whereas Psa seems to block bacterial uptake by interacting with a host receptor that doesn't direct internalization. The Deltacaf Deltapsa double mutant bound and invaded all three epithelial cell types well, revealing the presence of an undefined adhesin(s) and invasin(s).

Expression of Programmed Death–1 Ligand (PD‐L) 1, PD‐L2, B7‐H3, and Inducible Costimulator Ligand on Human Respiratory Tract Epithelial Cells and Regulation by Respiratory Syncytial Virus and Type 1 and 2 Cytokines

Respiratory syncytial virus (RSV) is associated with wheezing illness, and infections can occur repeatedly throughout life. We hypothesized that RSV infection of respiratory tract epithelial cells up-regulates B7 molecules that regulate memory immune responses and that type 1 and 2 cytokines differentially modulate this induction. We used flow-cytometric analysis to investigate programmed death-1 ligand (PD-L) 1, PD-L2, B7-H3, and inducible costimulatory ligand (ICOS-L) expression on tracheal (NCI-H292), bronchial (BEAS-2B), and alveolar (A549) epithelial cells; regulation of this expression by RSV, interferon (IFN)- gamma , and interleukin (IL)-4; and the effects of IFN-gamma and IL-4 on RSV-induced expression of these molecules. B7-H3 was strongly expressed, PD-L1 and ICOS-L were moderately expressed, and PD-L2 was weakly expressed on unstimulated tracheal, bronchial, and alveolar epithelial cells. RSV infection up-regulated PD-L1, PD-L2, and B7-H3 expression on all cells and ICOS-L expression on bronchial and alveolar epithelial cells. IL-4 treatment alone had no effect, whereas IFN-gamma treatment alone increased PD-L1 and PD-L2 expression on all cells and decreased B7-H3 expression on bronchial and alveolar epithelial cells. On RSV-infected alevolar epithelial cells, IFN-gamma treatment increased PD-L1 and PD-L2 expression and decreased B7-H3 and ICOS-L expression, and IL-4 treatment increased PD-L2 and B7-H3 expression and decreased ICOS-L expression. Respiratory tract epithelial cells express a wide range of B7 molecules. RSV infection increases their expression, and this expression is differentially regulated by IFN-gamma and IL-4. These processes may be involved in decreasing T cell antiviral immune responses to RSV and in RSV-associated wheezing.

Comparison of radiation sensitivity of rat respiratory tract epithelial cells

Comparison of radiation sensitivity of rat respiratory tract epithelial cells

Promoter Hypermethylation of p16 in Lung Tumors from Plutonium-exposed Rat and Respiratory Tract Epithelial Cell Lines

Promoter Hypermethylation of p16 in Lung Tumors from Plutonium-exposed Rat and Respiratory Tract Epithelial Cell Lines

Vitamin A supplementation for preventing morbidity and mortality in very low birthweight infants.

Vitamin A is necessary for normal lung growth and the ongoing integrity of respiratory tract epithelial cells. Preterm infants have low vitamin A status at birth and this has been associated with increased risk of developing chronic lung disease. Several studies have been undertaken to assess whether vitamin A supplementation beyond that routinely given in multivitamin preparations can reduce the incidence of this outcome. To assess the benefit of supplementation with vitamin A in very low birthweight infants. Searches were made of the Oxford Database of Perinatal Trials, MEDLINE, Cochrane Controlled Trials Register, and Science Citation Index. The reference lists of relevant trials, recent issues of paediatric and nutrition journals, abstracts and proceedings from relevant conferences in the English language were hand searched. Randomised controlled trials which compared the effects of supplemental vitamin A with standard vitamin A regimes in infants with birthweight </=1500g, and which reported clinical outcomes (death, chronic lung disease or bronchopulmonary dysplasia) and/or vitamin A concentrations were considered for the review. Data on mortality, requirement for supplemental oxygen at one month of age and at 36 weeks post-menstrual age, retinopathy of prematurity and nosocomial sepsis were excerpted by both reviewers independently. Data analysis was conducted according to the standards of the Cochrane Neonatal Review Group. Six eligible trials were identified, one having a much larger sample size than the others combined. The meta-analysis suggests supplementation with vitamin A results in benefit in terms of reducing oxygen requirement at 36 weeks post-menstrual age [summary RR 0.85 (0.73, 0.98), RD -8.5% (-15.9, -1.1), NNT 11.8 (6.3, 90.9)] and trends towards reduction in death or oxygen requirement at 36 weeks post-menstrual age [summary RR 0.89 (0.79, 1.00)], oxygen use in survivors at one month of age [summary RR 0.93 (0.86, 1.01)], and death or oxygen requirement at one month of age [summary RR 0.93 (0. 86, 1.00)]. Meta-analysis of the two studies which reported on retinopathy of prematurity suggests a trend towards reduced incidence in vitamin A supplemented infants. Supplementing very low birthweight infants with vitamin A is associated with a reduction in oxygen requirement amongst survivors at 36 weeks post-menstrual age. Whether clinicians decide to utilise repeat intramuscular doses of vitamin A to prevent chronic lung disease may depend upon the local incidence of this outcome and the value attached to achieving a modest reduction in this outcome, balanced against the lack of other proven benefits and the acceptability of treatment. The benefits, in terms of vitamin A status, safety and acceptability of delivering vitamin A in an intravenous emulsion compared with repeat intramuscular injections should be assessed in a further trial.

Mechanisms of neutrophil-induced DNA damage in respiratory tract epithelial cells.

Reactive oxygen species (ROS) released by neutrophils have been suggested to play an important role in cancer development. Since the mechanisms underlying this effect in the respiratory tract are still unclear, we evaluated DNA damage induced by neutrophils in respiratory tract epithelial cells in vitro and in vivo. For in vitro studies, rat lung epithelial cells (RLE) were co-incubated with activated neutrophils, neutrophil-conditioned medium, or hydrogen peroxide. For in vivo studies, we considered the human nose as a target organ, comparing neutrophilic inflammation in the nasal lavage fluid with the oxidative DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in epithelial cells obtained by nasal brush. Our in vitro data show that human neutrophils are able to induce both 8-OHdG and strand breaks in DNA from RLE cells. Our data also suggest that DNA damage induced by neutrophils is inhibited when neutrophil-derived H2O2 is consumed by myeloperoxidase. In contrast, in the nose no association between neutrophil numbers and 8-OHdG was found. Therefore, it remains unclear whether neutrophils pose a direct genotoxic risk for the respiratory tract epithelium during inflammation, andmore in vivo studies are needed to elucidate the possible association between neutrophils and genotoxicity in the lung.

Adherence of Burkholderia cepacia to respiratory tract epithelial cells and inhibition with dextrans

Adherence of Burkholderia cepacia to cells of the respiratory tract of patients with cystic fibrosis (CF) appears to be a necessary precondition for colonization and infection. To date, no effective anti-adhesive strategy has been devised for preventing B. cepacia infection in CF patients. It was found in this study that B. cepacia adhered to respiratory epithelial cells both in vitro and in vivo. However, strains with cable-like pili (Cbl) exhibited the typical clump formation on pneumocytes, whereas non-cable piliated strains predominantly showed single-cell adherence. Dextrans (nominally 4000-10000 Da) significantly inhibited adhesion of B. cepacia to A549 pneumocytes. When compared on an equal weight basis, the nominally 10000 Da dextran was most inhibitory. A dose-dependent inhibitory effect (up to 80 mg ml(-1)) was observed for most strains. Dextran exerted less of an anti-adhesive effect on the two Cbl+ strains than on the others which were Cbl-. Dextrans appeared to block the adherence in a non-specific fashion, as shown by the observations that the inhibitory effect was readily reversible and oligosaccharides composed of 2-4 glucose units with the same alpha-1,6 linkage were not inhibitory. The mean molecular masses of dextrans used in this study, as determined by gel filtration and MS, were approximately 10-fold lower than those indicated by the manufacturers. Our data suggest that dextran of nominal molecular mass 4000 Da at a concentration of 40 mg ml(-1) (10 mM according to manufacturer's quoted molecular mass) or more may be useful in patients with CF to prevent colonization and infection with B. cepacia.

Invasion of murine respiratory epithelial cells in vivo by Burkholderia cepacia.

Pulmonary infections caused by Burkholderia cepacia are an important cause of morbidity and mortality in cystic fibrosis (CF) patients. Several features suggestive of invasion and intracellular sequestration of B. cepacia in CF are persistence of infection in the face of antibiotic therapy and a propensity to cause bacteraemic infections in patients with CF. A mouse respiratory challenge model was used to investigate the invasion phenotype of B. cepacia in vivo. After intratracheal inoculation, epidemic B. cepacia strains translocated from lung to liver and spleen; however, all bacteria were cleared from all organs within 7 days. B. cepacia strains, irrespective of cable piliation, were capable of attaching to and then invading murine respiratory tract epithelial cells. Histopathological examination of lungs showed interstitial infiltrates comprised mainly of polymorphonuclear leucocytes and were associated with widened alveolar septa. Electron microscopy demonstrated B. cepacia within epithelial cells and pulmonary macrophages. This study provides support for in-vitro observations that B. cepacia strains from patients with CF adhere to and then invade respiratory epithelial cells. The invasion phenotype in B. cepacia may be an important virulence factor in CF infections.

Phagocyte oxidants and nitric oxide in cystic fibrosis: new therapeutic targets?

Chronic persistent respiratory tract (RT) infection and overly exuberant activation of host inflammatory-immune processes represent the paramount pathobiologic consequences of cystic fibrosis (CF). The host inflammatory-immune system activation includes the production of reactive oxygen species (ROS) and nitric oxide (NO*), which are helpful in RT antimicrobial defenses but under conditions of excess are believed to be harmful to host tissues. However, the genetic defect underlying CF is recognized to uniquely affect inflammatory-immune (oxidative) processes, including alterations in cytokine release, phagocyte activation, antioxidant mechanisms, and NO* synthesis and metabolism, and these appear to contribute importantly to the persistent inflammation and infection in patients with CF. The dysregulation of inflammatory-immune responses has also been observed in CF epithelial cells and in some mouse models of CF, and marked advances in our understanding of these processes can be expected from future studies in such model systems. The emerging ideas of how ROS may influence molecular events that control inflammatory protease/antiprotease homeostasis and RT epithelial cell signaling and gene expression, and the role that augmentation of local antioxidant micronutrients (aerosolized and/or systemically administrated) might play in these effects, still remain to be further established and clarified. Further understanding of these processes can be expected to play an important role in future treatment directed toward the devastating effects of CF on the RT of patients with this disorder.

Group B streptococci and other gram-positive cocci bind to cytokeratin 8.

Group B streptococci (GBS) adhere to surface receptors present on epithelial cells; these receptors include fibronectin and laminin. To identify other possible receptors, plasma membranes from A549 cells, a respiratory tract epithelial cell line, were prepared. These plasma membranes were tested in a protein blot analysis using radiolabeled GBS as a probe. GBS adhered to two species, with molecular masses of 50 kDa (p50) and 57 kDa (p57). We concluded that p50 and p57 correspond to two forms of cytokeratin 8 (CK8) on the basis of the following results: (i) protein blot results demonstrated that p50 and p57 exactly comigrated with two forms of CK8 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE); (ii) p50 and p57 exactly comigrated with CK8 after separation by two-dimensional PAGE; (iii) CK8 in solution bound to GBS, as demonstrated by immunoblot analysis of proteins from A549 lysates that bound to GBS in a liquid-phase assay; and (iv) radiolabeled GBS bound to A549 lysate-derived CK8 that had been captured in anti-CK8-coated microtiter wells. CK8 bound to COH1-13, an acapsular mutant of COH1, demonstrating that adherence is not mediated by capsular polysaccharide. Trypsin-treated GBS did not bind to CK8, indicating that adherence is mediated via a protein on the surface of GBS. Soluble CK8 bound to six of six GBS strains tested. Soluble CK8 also bound to Staphylococcus aureus, Lactococcus lactis, Enterococcus faecalis, and Streptococcus pyogenes. We hypothesize that adherence of GBS to cytokeratin may be important for maintenance of colonization at sites of keratinized epithelium, such as the vagina, or for adherence of these bacteria to damaged epithelial cells at other sites.

Nitrite-induced deamination and hypochlorite-induced oxidation of DNA in intact human respiratory tract epithelial cells

No modification of purine or pyrimidine bases was observed when isolated DNA was incubated with 1 mM nitrite at pH 7.4. However, exposure of human bronchial epithelial cells in culture medium at pH 7.4 to nitrite at concentrations of 100 μM or greater led to deamination of purine bases in cellular DNA. Deamination was more extensive in cells exposed to lower extracellular pH values and higher nitrite concentrations. Significant increases in the levels of xanthine and hypoxanthine, putative deamination products of guanine and adenine, respectively, were observed in DNA from nitrite-treated cells but no rise in any base oxidation products such as 8-hydroxyguanine. This pattern of damage suggests that exposure of cells to nitrite (even at pH 7.4) leads to intracellular generation of “reactive nitrogen species” capable of deaminating purines in DNA. In addition, significant DNA strand breakage occurred in nitrite-treated cells. The time course of base damage suggested that the repair of deaminated purine lesions in these cells is slow. By contrast, DNA isolated from cells exposed to hypochlorous acid (HOCl) has significant oxidation of pyrimidine bases and chlorination of cytosine but little oxidation of purines. Exposure of cells to both species (NO 2 − plus HOCl) potentiated the oxidative DNA base damage observed but decreased the extent of deamination. We hypothesize that this is due to the formation of nitryl chloride (NO 2Cl) from reaction of HOCl with •NO 2 −. The relevance of our observations to events in the stomach and respiratory tract, at sites of inflammation, and in ischemic tissues is discussed.