Primary human airway epithelial cell-dependent inhibition of human lung mast cell degranulation

Introduction

Chronic mast cell activation is a characteristic feature of asthma. BEAS-2B human airway epithelial cells (AEC) profoundly inhibit both constitutive and IgE-dependent human lung mast cell (HLMC) histamine release. The aim of this study was to examine the regulation of HLMC degranulation by primary AEC from healthy and asthmatic subjects, and investigate further the inhibitory mechanism.

Methods

HLMC were co-cultured with both BEAS-2B and primary AEC grown as monolayers or air-liquid interface (ALI) cultures.

Results

Both constitutive and IgE-dependent HLMC histamine release were attenuated by BEAS-2B, primary AEC monolayers and ALI cultures. This occurred in the absence of HLMC-AEC contact indicating the presence of a soluble factor. Unlike healthy ALI AEC, asthmatic ALI-AEC did not significantly reduce constitutive histamine release. AEC inhibitory activity was transferable in primary AEC monolayer supernatant, but less active than with Transwell co-culture, suggesting that the inhibitory factor was labile. The AEC inhibitory effects were attenuated by both AEC wounding and pertussis toxin, indicating the involvement of a G₀/G_i receptor coupled mechanism. Solid phase extraction of lipids (<10 kDa) removed the AEC inhibitory activity. The lipid derivatives resolvin D1 and D2 and lipoxin A₄ attenuated HLMC histamine release in a dose-dependent fashion but were not detectable in co-culture supernatants.

Conclusions

Primary AEC suppress HLMC constitutive and IgE-dependent histamine secretion through the release of a soluble, labile lipid mediator(s) that signals through the G₀/G_i receptor coupled mechanism. Manipulation of this interaction may have a significant therapeutic role in asthma.     

Sea urchin Hox genes: insights into the ancestral Hox cluster

We describe the Hox cluster in the radially symmetric sea urchin and compare our findings to what is known from clusters in bilaterally symmetric animals. Several Hox genes from the direct-developing sea urchin Heliocidaris erythrogramma are described. CHEF gel analysis shows that the Hox genes are clustered on a < or = 300 kilobase (kb) fragment of DNA, and only a single cluster is present, as in lower chordates and other nonvertebrate metazoans. Phylogenetic analyses of sea urchin, amphioxus, Drosophila, and selected vertebrate Hox genes confirm that the H. erythrogramma genes, and others previously cloned from other sea urchins, belong to anterior, central, and posterior groups. Despite their radial body plan and lack of cephalization, echinoderms retain at least one of the anterior group Hox genes, an orthologue of Hox3. The structure of the echinoderm Hox cluster suggests that the ancestral deuterostome had a Hox cluster more similar to the current chordate cluster than was expected Sea urchins have at least three Abd-B type genes, suggesting that Abd-B expansion began before the radiation of deuterostomes.      

Smoking cessation and bronchial epithelial remodelling in COPD: a cross-sectional study

Background

Chronic Obstructive Pulmonary Disease (COPD) is associated with bronchial epithelial changes, including squamous cell metaplasia and goblet cell hyperplasia. These features are partially attributed to activation of the epidermal growth factor receptor (EGFR). Whereas smoking cessation reduces respiratory symptoms and lung function decline in COPD, inflammation persists. We determined epithelial proliferation and composition in bronchial biopsies from current and ex-smokers with COPD, and its relation to duration of smoking cessation.

Methods

114 COPD patients were studied cross-sectionally: 99 males/15 females, age 62 ± 8 years, median 42 pack-years, no corticosteroids, current (n = 72) or ex-smokers (n = 42, median cessation duration 3.5 years), postbronchodilator FEV₁ 63 ± 9% predicted. Squamous cell metaplasia (%), goblet cell (PAS/Alcian Blue⁺) area (%), proliferating (Ki-67⁺) cell numbers (/mm basement membrane), and EGFR expression (%) were measured in intact epithelium of bronchial biopsies.

Results

Ex-smokers with COPD had significantly less epithelial squamous cell metaplasia, proliferating cell numbers, and a trend towards reduced goblet cell area than current smokers with COPD (p = 0.025, p = 0.001, p = 0.081, respectively), but no significant difference in EGFR expression. Epithelial features were not different between short-term quitters (<3.5 years) and current smokers. Long-term quitters (≥3.5 years) had less goblet cell area than both current smokers and short-term quitters (medians: 7.9% vs. 14.4%, p = 0.005; 7.9% vs. 13.5%, p = 0.008; respectively), and less proliferating cell numbers than current smokers (2.8% vs. 18.6%, p < 0.001).

Conclusion

Ex-smokers with COPD had less bronchial epithelial remodelling than current smokers, which was only observed after long-term smoking cessation (>3.5 years).

Trial registration

NCT00158847     

Immunocompetence increases with larval body size in a phytophagous moth

Despite the obvious benefit of an immune system, its efficacy against pathogens and parasites may show great variation among individuals, populations and species. Understanding the causes of this variation is becoming a central theme in ecology. Many biotic and abiotic factors are known to influence immunocompetence (temperature, age, etc.). However, for a given age, size among individuals varies, probably as a result of accumulated resources. Thus, these variable resources could be allocated to immune defence and, consequently, body size may explain part of the variation in immune responsiveness. However, the influence of body size on immune defence is often overlooked. The present study investigates variations in haemocyte count and phenoloxidase activity in larvae of the phytophagous vine moth Eupoecilia ambiguella Hübner of the same age, although differing in body size. The measurements of immune function are made both when the insects are immunologically naïve and 24 h after a bacterial immune challenge. The base levels of these immune parameters do not covary with body size in naïve larvae. After the bacterial immune challenge, more haemocytes and phenoloxidase enzyme are mobilized, and the mobilization of these immune effectors is correlated positively with individual body size. Thus, larger larvae exhibit higher immunocompetence than smaller ones, suggesting that smaller larvae might be more vulnerable to infection. These results suggest that body size is probably an underestimated variable, which nevertheless modulates the insect immune system and should thus be considered as a covariate in insect immune system measurement. It is recommended therefore, that body size should be taken into account in ecological immunity studies with insects. © 2013 The Royal Entomological Society     

Caspase-mediated cleavage of the stacking protein GRASP65 is required for Golgi fragmentation during apoptosis

The mammalian Golgi complex is comprised of a ribbon of stacked cisternal membranes often located in the pericentriolar region of the cell. Here, we report that during apoptosis the Golgi ribbon is fragmented into dispersed clusters of tubulo-vesicular membranes. We have found that fragmentation is caspase dependent and identified GRASP65 (Golgi reassembly and stacking protein of 65 kD) as a novel caspase substrate. GRASP65 is cleaved specifically by caspase-3 at conserved sites in its membrane distal COOH terminus at an early stage of the execution phase. Expression of a caspase-resistant form of GRASP65 partially preserved cisternal stacking and inhibited breakdown of the Golgi ribbon in apoptotic cells. Our results suggest that GRASP65 is an important structural component required for maintenance of Golgi apparatus integrity.     

Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport

The fate of tetanus toxin (mol wt 150,000) subsequent to its retrograde axonal transport in peripheral sympathetic neurons of the rat was studied by both electron microscope autoradiography and cytochemistry using toxin-horseradish peroxidase (HRP) coupling products, and compared to that of nerve growth factor (NGF), cholera toxin, and the lectins wheat germ agglutinin (WGA), phytohaemagglutinin (PHA), and ricin. All these macromolecules are taken up by adrenergic nerve terminals and transported retrogradely in a selective, highly efficient manner. This selective uptake and transport is a consequence of the binding of these macromolecules to specific receptive sites on the nerve terminal membrane. All these ligands are transported in the axons within smooth vesicles, cisternae, and tubules. In the cell bodies these membrane compartments fuse and most of the transported macromolecules are finally incorporated into lysosomes. The cell nuclei, the parallel golgi cisternae, and the extracellular space always remain unlabeled. In case the tetanus toxin, however, a substantial fraction of the labeled material appears in presynaptic cholinergic nerve terminals which innervate the labeled ganglion cells. In these terminals tetanus toxin-HRP is localized in 500-1,000 A diam vesicles. In contrast, such a retrograde transsynaptic transfer is not at all or only very rarely detectable after retrograde transport of cholera toxin, NGF, WGA, PHA, or ricin. An atoxic fragment of the tetanus toxin, which contains the ganglioside-binding site, behaves like intact toxin. With all these macromolecules, the extracellular space and the glial cells in the ganglion remain unlabeled. We conclude that the selectivity of this transsynaptic transfer of tetanus toxin is due to a selective release of the toxin from the postsynaptic dendrites. This release is immediately followed by an uptake into the presynaptic terminals.     

Biogenesis of the sorting endosome: the role of Rab5

Rab5 is a regulatory guanosine triphosphatase that is associated with the sorting endosome and participates in endosomal membrane fusion reactions. Recent experiments have provided insights into Rab5 function by demonstrating direct links between Rab5-interacting proteins and components of the membrane fusion apparatus. In addition, a realisation that Rab5 has additional functions in endosome biogenesis is emerging. These advances may be profoundly important in changing the way that we view the sorting endosome and in developing models that properly reflect the dynamic qualities of the endocytic pathway.     

Colposcopy services in the West Midlands region.

A survey of all 72 consultant gynaecologists in the West Midlands region was carried out to determine their views on colposcopy services. All districts provided a colposcopy service and 47 consultants practised colposcopy. The consultants differed considerably in their views on criteria for referring women for investigation after smears. All but one thought that a positive smear result was an indication for immediate referral but whereas 55 thought that women with one or more inflammatory smear should be referred, 17 did not believe this to be necessary. Sixty seven consultants thought a preoperative colposcopic assessment desirable, but 10 had to ration referrals because of limited resources. Four forms of treatment were used: 17 districts had a carbon dioxide laser, eight used low voltage diathermy loop excision, two had only a cold coagulator, and one used only cold cautery. There was widespread dissatisfaction with resources and about the increasing amount of time spent on the service at the expense of other work. Consultants were also concerned about the relevance of much of what was done to the aim of the screening programme--reducing mortality from cervical cancer. Although additional resources are needed in some districts, referral policies, indications for colposcopic assessment, training, choice of treatment, and the value of follow up need to be reviewed if the service is to be improved.