Diesel exhaust particulate matter induces multinucleate cells and zinc transporter-dependent apoptosis in human airway cells

The cellular effects of biodiesel emissions particulate matter (BDEP) and petroleum diesel emissions particulate matter (PDEP) were compared using a human airway cell line, A549. At concentrations of 25 µg/ml, diesel particulate matter induced the formation of multinucleate cells. In cells treated with a mixture of 80% PDEP:20% BDEP, 52% of cells were multinucleate cells compared with only 16% of cells treated with 20% PDEP:80% BDEP with a background multinucleate rate of 7%. These results demonstrate a causal relation between the formation of multinucleate cells and exposure to exhaust particulate matter, in particular diesel exhaust. Exposure of A549 cells to PDEP induced apoptosis, seen by active caspase-3 expression and the presence of cleaved pancytokeratin. PDEP exhaust was a much stronger inducer of cellular death through apoptosis than BDEP. There was an eightfold increase in the expression of SLC30A3 (zinc transporter-3 or ZnT3) in cells exposed to 80% PDEP:20% BDEP compared to untreated cells. The increase in ZnT3 expression seen in apoptotic cells following PDEP suggests a role for this zinc transporter in the apoptotic pathway, possibly through controlling zinc fluxes. As exposure to diesel exhaust particles is associated with asthma and apoptosis in airway cells, diesel exhaust particles may directly contribute to asthma by inducing epithelial cell death through apoptotic pathway.

Bovine milk fat enriched in conjugated linoleic and vaccenic acids attenuates allergic airway disease in mice

Background: It has been argued that a reduction in the Western diet of anti-inflammatory unsaturated lipids, such as n-3 polyunsaturated fatty acids, has contributed to the increase in the frequency and severity of allergic diseases.

Objective: We investigated whether feeding milk fat enriched in conjugated linoleic acid and vaccenic acids (VAs) ('enriched' milk fat), produced by supplementing the diet of pasture-fed cows with fish and sunflower oil, will prevent development of allergic airway responses.

Methods: C57BL/6 mice were fed a control diet containing soybean oil and diets supplemented with milk lipids. They were sensitized by intraperitoneal injection of ovalbumin (OVA) on days 14 and 28, and challenged intranasally with OVA on day 42. Bronchoalveolar lavage fluid, lung tissues and serum samples were collected 6 days after the intranasal challenge.

Results: Feeding of enriched milk fat led to marked suppression of airway inflammation as evidenced by reductions in eosinophilia and lymphocytosis in the airways, compared with feeding of normal milk fat and control diet. Enriched milk fat significantly reduced circulating allergen-specific IgE and IgG1 levels, together with reductions in bronchoalveolar lavage fluid of IL-5 and CCL11. Treatment significantly inhibited changes in the airway including airway epithelial cell hypertrophy, goblet cell metaplasia and mucus hypersecretion. The two major components of enriched milk fat, cis-9, trans-11 conjugated linoleic acid and VA, inhibited airway inflammation when fed together to mice, whereas alone they were not effective.

Conclusion: Milk fat enriched in conjugated linoleic and VAs suppresses inflammation and changes to the airways in an animal model of allergic airway disease.

An aqueous Chlorella extract inhibits IL-5 production by mast cells in vitro and reduces ovalbumin-induced eosinophil infiltration in the airway in mice in vivo

An aqueous extract of the edible microalga, Chlorella pyrenoidosa (CP) (1), has recently been tested for its immunomodulatory effects in a human clinical trial. Here, the CP extract was dialyzed and fractionated using Sephadex G 100 chromatography. The effects of a dialyzed aqueous CP extract, fraction 2, on mast cell mediator release in vitro and ovalbumin-induced allergic airway inflammation in vivo were examined. In vitro, treatment of mouse bone marrow-derived mast cells with 2 for 18 h significantly inhibited antigen (trinitrophenyl-BSA)-induced IL-5 production. In vivo, treatment of mice with 2 during ovalbumin sensitization and stimulation process significantly reduced eosinophil and neutrophil infiltration in the airways. Moreover, fractions obtained by size exclusion chromatography of 2 inhibited IgE-dependent cytokine GM-CSF production from human cord blood-derived mast cells. Taken together, these results suggest that 2 is composed of biopolymers with anti-allergic potential.

c-Cbl facilitates endocytosis and lysosomal degradation of cystic fibrosis transmembrane conductance regulator in human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl− channel expressed in the apical membrane of fluid-transporting epithelia. The apical membrane density of CFTR channels is determined, in part, by endocytosis and the postendocytic sorting of CFTR for lysosomal degradation or recycling to the plasma membrane. Although previous studies suggested that ubiquitination plays a role in the postendocytic sorting of CFTR, the specific ubiquitin ligases are unknown. c-Cbl is a multifunctional molecule with ubiquitin ligase activity and a protein adaptor function. c-Cbl co-immunoprecipitated with CFTR in primary differentiated human bronchial epithelial cells and in cultured human airway cells. Small interfering RNA-mediated silencing of c-Cbl increased CFTR expression in the plasma membrane by inhibiting CFTR endocytosis and increased CFTR-mediated Cl− currents. Silencing c-Cbl did not change the expression of the ubiquitinated fraction of plasma membrane CFTR. Moreover, the c-Cbl mutant with impaired ubiquitin ligase activity (FLAG-70Z-Cbl) did not affect the plasma membrane expression or the endocytosis of CFTR. In contrast, the c-Cbl mutant with the truncated C-terminal region (FLAG-Cbl-480), responsible for protein adaptor function, had a dominant interfering effect on the endocytosis and plasma membrane expression of CFTR. Moreover, CFTR and c-Cbl co-localized and co-immunoprecipitated in early endosomes, and silencing c-Cbl reduced the amount of ubiquitinated CFTR in early endosomes. In summary, our data demonstrate that in human airway epithelial cells, c-Cbl regulates CFTR by two mechanisms: first by acting as an adaptor protein and facilitating CFTR endocytosis by a ubiquitin-independent mechanism, and second by ubiquitinating CFTR in early endosomes and thereby facilitating the lysosomal degradation of CFTR.

Apical localization of zinc transporter ZnT4 in human airway epithelial cells and its loss in a murine model of allergic airway inflammation

The apical cytoplasm of airway epithelium (AE) contains abundant labile zinc (Zn) ions that are involved in the protection of AE from oxidants and inhaled noxious substances. A major question is how dietary Zn traffics to this compartment. In rat airways, in vivo selenite autometallographic (Se-AMG)-electron microscopy revealed labile Zn-selenium nanocrystals in structures resembling secretory vesicles in the apical cytoplasm. This observation was consistent with the starry-sky Zinquin fluorescence staining of labile Zn ions confined to the same region. The vesicular Zn transporter ZnT4 was likewise prominent in both the apical and basal parts of the epithelium both in rodent and human AE, although the apical pools were more obvious. Expression of ZnT4 mRNA was unaffected by changes in the extracellular Zn concentration. However, levels increased 3-fold during growth of cells in air liquid interface cultures and decreased sharply in the presence of retinoic acid. When comparing nasal versus bronchial human AE cells, there were significant positive correlations between levels of ZnT4 from the same subject, suggesting that nasal brushings may allow monitoring of airway Zn transporter expression. Finally, there were marked losses of both basally-located ZnT4 protein and labile Zn in the bronchial epithelium of mice with allergic airway inflammation. This study is the first to describe co-localization of zinc vesicles with the specific zinc transporter ZnT4 in airway epithelium and loss of ZnT4 protein in inflamed airways. Direct evidence that ZnT4 regulates Zn levels in the epithelium still needs to be provided. We speculate that ZnT4 is an important regulator of zinc ion accumulation in secretory apical vesicles and that the loss of labile Zn and ZnT4 in airway inflammation contributes to AE vulnerability in diseases such as asthma.

PPARγ ligands, 15-deoxy-Δ12,14-prostaglandin J2 and rosiglitazone regulate human cultured airway smooth muscle proliferation through different mechanisms

1. The influence of two peroxisome proliferator-activated receptor γ (PPARγ) ligands, a thiazolidinedione, rosiglitazone (RG) and the prostaglandin D2 metabolite 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) on the proliferation of human cultured airway smooth muscle (HASM) was examined.

2. The increases in HASM cell number in response to basic fibroblast growth factor (bFGF, 300 pm) or thrombin (0.3 U ml−1) were significantly inhibited by either RG (1–10 μm) or 15d-PGJ2 (1–10 μm). The effects of RG, but not 15d-PGJ2, were reversed by the selective PPARγ antagonist GW9662 (1 μm).

3. Neither RG nor 15d-PGJ2 (10 μm) decreased cell viability, or induced apoptosis, suggesting that the regulation of cell number was due to inhibition of proliferation, rather than increased cell death.

4. Flow-cytometric analysis of HASM cell cycle distribution 24 h after bFGF addition showed that RG prevented the progression of cells from G1 to S phase. In contrast, 15d-PGJ2 caused an increase in the proportion of cells in S phase, and a decrease in G2/M, compared to bFGF alone.

5. Neither RG nor 15d-PGJ2 inhibited ERK phosphorylation measured 6 h post mitogen addition. The bFGF-mediated increase in cyclin D1 protein levels after 8 h was reduced in the presence of 15d-PGJ2, but not RG.

6. Although both RG and 15d-PGJ2 can inhibit proliferation of HASM irrespective of the mitogen used, only the antiproliferative effects of RG appear to be PPARγ-dependent. The different antimitogenic mechanisms of 15d-PGJ2 and synthetic ligands for PPARγ may be exploited to optimise the potential for these compounds to inhibit airway remodelling in asthma.

Enhanced cytotoxic function of natural killer and natural killer T-like cells associated with decreased CD94 (Kp43) in the chronic obstructive pulmonary disease airway

Background and objective: Natural killer (NK) and natural killer T (NKT)-like cells represent a small but important proportion of effector lymphocytes that we have previously shown to be major sources of pro-inflammatory cytokines and granzymes. We hypothesized that these cells would be increased in the airway in chronic obstructive pulmonary disease (COPD), accompanied by reduced expression of the inhibitory receptor CD94 (Kp43) and increased expression of cytotoxic mediators granzyme B and perforin.
Methods: We measured NK and NKT-like cells and their expression of CD94 in the blood of COPD patients (n = 71; 30 current and 41 ex-smokers), smokers (16) and healthy controls (25), and bronchoalveolar lavage fluid (BALF) from a cohort of subjects (19 controls, 12 smokers, 33 COPD). Activation was assessed by measuring CD69 in blood and the cytotoxic potential of NK cells by measuring granzymes A and B, and using a cytotoxicity assay in blood and BALF.
Results: In blood in COPD, there were no significant changes in the proportion of NK or NKT-like cells or expression of granzyme A or NK cytotoxic potential versus controls. There was, however, increased expression of granzyme B and decreased expression of CD94 by both cell types versus controls. The proportion of NK and NKT-like cells were increased in BALF in COPD, associated with increased NK cytotoxicity, increased expression of granzyme B and decreased expression of the inhibitory receptor CD94 by both cell types.
Conclusions: Treatment strategies that target NK and NKT-like cells, their cytotoxicity and production of inflammatory mediators in the airway may improve COPD morbidity.

Airway clearance of apoptotic cells in COPD

Apoptosis of bronchial epithelial cells and the phagocytic clearance of these cells by alveolar macrophages (a process termed efferocytosis) are integral processes leading to repair of airway epithelial injury. Efferocytosis allows for the removal of apoptotic material with minimal inflammation and prevents the development of secondary necrosis and ongoing inflammation. Defective efferocytosis and the increased presence of apoptotic cells have been identified in the airways of subjects with chronic obstructive pulmonary disease (COPD). There are three major potential causes for this accumulation of apoptotic cells: (i) increased apoptosis per se as a result of an increase in apoptotic mediators, (ii) defects in the recognition of apoptotic cells by AM and (iii) failure to clear the unwanted cells by the process of efferocytosis. The implications of these processes in COPD and novel treatment strategies aimed at improving clearance of apoptotic cells form the focus of the present review.

Best practice in stabilisation of oral endotracheal tubes : a systematic review

Mechanical ventilation of patients in intensive care units is common practice. Artificial airways are utilised to facilitate ventilation and the endotracheal tube (ETT) is most commonly used for this purpose. The ETT must be stabilised to optimise ventilation and avoid displacement or unplanned extubation. Tube movement is a major factor in causing airway trauma. A destabilised tube can cause fatal complications. A systematic review was conducted to identify and analyse the best available evidence on ETT stabilisation to determine which stabilisation method resulted in reduced tube displacement and the least amount of unplanned or accidental extubations. The types of stabilisations included one or a combination of the following methods: twill or cotton tape, adhesive tape, gauze, or a manufactured device. All relevant randomised controlled and quasi-experimental studies of ETT stabilisation practices, identified through electronic and hand searching, were assessed for inclusion in the study. One published randomised controlled trial and six published quasi-experimental studies met the inclusion and exclusion criteria and were retrieved. Data were extracted independently by two reviewers. Results of the systematic review showed that no single method of ETT stabilisation could be identified as superior for minimising tube displacement and unplanned or accidental extubations. Rigorous randomised controlled trials with clearly identified and described ETT stabilisation methods are required to establish best practice. In addition, comparative research to evaluate cost effectiveness and nursing time requirements would also be of significant benefit to critical care nursing practice.

Biodiesel breathes better

It is long-established that car exhaust fumes cause respiratory disease, and more recently the particulate matter in diesel exhaust has been implicated in the death of human airway cells.  However, new research reveals that biodiesel is a safer alternative.

Tracheostomy decannulation failure rate following critical illness : a prospective descriptive study

Background : Tracheostomy is a well established and practical approach to airway management for patients requiring extended periods of mechanical ventilation or airway protection. Little evidence is available to guide the process of weaning and optimal timing of tracheostomy tube removal. Thus, decannulation decisions are based on clinical judgement. The aim of this study was to describe decannulation practice and failure rates in patients with tracheostomy following critical illness.

Methods : A prospective descriptive study was conducted of consecutive patients who received a tracheostomy at a tertiary metropolitan public hospital intensive care unit (ICU) between March 2002 and December 2006. Data were analysed using descriptive and inferential tests.

Results : Of the 823 decannulation decisions, there were 40 episodes of failed decannulation, a failure rate of 4.8%. These 40 episodes occurred in 35 patients: 31 patients failed once, 3 patients failed twice and 1 patient failed three times. There was no associated mortality. Simple stoma recannulation was required in 25 episodes, with none of these patients readmitted to ICU. Translaryngeal intubation and readmission to ICU took place for the remaining 15 episodes. The primary reason for decannulation failure was sputum retention. Twenty-four patients (60%) failed decannulation within 24 h, with 14 of these occurring within 4 h.

Conclusions : Clinical assessments coupled with professional judgement to decide the optimal time to remove tracheostomy tubes in patients following critical illness resulted in a failure rate comparable with published data. Although reintubation and readmission to ICU was required in just over one third of failed decannulation episodes, there was no associated mortality or other significant adverse events. Our data suggest nurses need to exercise high levels of clinical vigilance during the first 24 h following decannulation, particularly the first 4 h to detect early signs of respiratory compromise to avoid adverse outcomes.

Compositions of cis-9, trans-11 conjugated linoleic acid and vaccenic acid and uses thereof

The invention relates to use of the cis-9, trans-11 isomer of conjugated linoleic acid or a salt or ester thereof (cis-9, trans-11 CLA) and vaccenic acid or a salt or ester thereof (VA) to treat or prevent conditions associated with one or more of leukocyte infiltration, eosinophilia, airway remodelling, bronchoconstriction, mucus hypersecretion, and lung and skin inflammation. The present invention also relates to a composition comprising cis-9, trans-11 CLA and VA and use of the composition to treat or pre-vent conditions associated with one or more of leukocyte infiltration, eosinophilia, airway remodelling, bronchoconstriction, mucus hypersecretion, and lung and skin inflammation. In particular, the medicinal uses, compositions and methods of the invention may be used to treat or prevent conditions such as asthma and dermatitis, and related disorders.

c-9 t-11 conjugated linoleic acid-enriched milk fat and uses thereof

Disclosed is the use of milk fat enriched with c-9, t-l l conjugated linoleic acid (CLA) or a salt, ester or precursor thereof for treating or preventing a condition associated with one or more of leukocyte infiltration, eosinophilia, IgE secretion, airway remodelling, bronchoconstriction and mucus hypersecretion.

CLA-enriched milkfat and uses thereof

The present invention relates to use of c-9, t-11 CLA or a salt, ester or precursor thereof or CLA-enriched milk fat comprising milk fat enriched with c-9, t-11 CLA or a salt, ester or precursor thereof for treating or preventing conditions such as those associated with one or more of leukocyte infiltration, eosinophilia, IgE secretion, airway remodelling, bronchoconstriction and mucus hypersecretion. The invention also relates to a pharmaceutical composition comprising CLA-enriched milk fat.

Identification and possible disease mechanisms of an under-recognized fungus, Aureobasidium pullulans

Background: Investigations into the occurrence and health effects of yeast-like fungi in the outdoor air in the US have been limited. We sought to identify a respirable-sized fungus common in the Pasadena air, locate a major source for the emissions and investigate its relevance to allergic disease. Methods: Yeast-like fungi sampled from the environment were isolated, microscopically examined and sequenced. Pasadena allergy patients were skin tested with commercially available fungal extracts. Patient serum was immunoanalyzed for specific IgE reactivity. Nearby vegetation was analyzed in a controlled emission chamber to find a major source for the aerosols. Results: Hyaline unicellular conidia comprised up to 90% (41,250 m^-3 of air) of total fungal counts and generally peaked at night and during periods of rainfall and ensuing winds throughout the fall and winter. Flowers were determined to be a major source of the emissions. The cellular and colonial morphology of isolates were consistent with Aureobasidium species. The sequence of the D1/D2 region of the 26S ribosomal subunit of isolates from flowers showed identity to two strains of Aureohasidium pullulans (black yeast). Seventeen percent (16/94) of atopic individuals had positive skin testing with A. pullulans extract. Patient sera gE identified several high molecular weight allergens in Aureobasidium extracts. Conclusions: Respirable-sized conidia of A. pullulans are emitted from flowers and form high concentrations in the air. They are associated with immediate reactivity on skin tests, bind to patient sera IgE, and might be relevant in allergic upper and lower airway diseases.