Ann Dewar

Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I

The execution phase of apoptosis is characterized by marked changes in cell morphology that include contraction and membrane blebbing. The actin–myosin system has been proposed to be the source of contractile force that drives bleb formation, although the biochemical pathway that promotes actin–myosin contractility during apoptosis has not been identified. Here we show that the Rho effector protein ROCK I, which contributes to phosphorylation of myosin light-chains, myosin ATPase activity and coupling of actin–myosin filaments to the plasma membrane, is cleaved during apoptosis to generate a truncated active form. The activity of ROCK proteins is both necessary and sufficient for formation of membrane blebs and for re-localization of fragmented DNA into blebs and apoptotic bodies.

Mutations in CCDC39 and CCDC40 are the major cause of primary ciliary dyskinesia with axonemal disorganization and absent inner dynein arms.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder caused by cilia and sperm dysmotility. About 12% of cases show perturbed 9+2 microtubule cilia structure and inner dynein arm (IDA) loss, historically termed “radial spoke defect.” We sequenced CCDC39 and CCDC40 in 54 “radial spoke defect” families, as these are the two genes identified so far to cause this defect. We discovered biallelic mutations in a remarkable 69% (37/54) of families, including identification of 25 (19 novel) mutant alleles (12 in CCDC39 and 13 in CCDC40). All the mutations were nonsense, splice, and frameshift predicting early protein truncation, which suggests this defect is caused by “null” alleles conferring complete protein loss. Most families (73%; 27/37) had homozygous mutations, including families from outbred populations. A major putative hotspot mutation was identified, CCDC40 c.248delC, as well as several other possible hotspot mutations. Together, these findings highlight the key role of CCDC39 and CCDC40 in PCD with axonemal disorganization and IDA loss, and these genes represent major candidates for genetic testing in families affected by this ciliary phenotype. We show that radial spoke structures are largely intact in these patients and propose this ciliary ultrastructural abnormality be referred to as “IDA and microtubular disorganisation defect,” rather than “radial spoke defect.”

Twenty-year review of quantitative transmission electron microscopy for the diagnosis of primary ciliary dyskinesia

Background The examination of ciliary ultrastructure in a nasal sample remains a definitive diagnostic test for primary ciliary dyskinesia (PCD). Methods The quantitative assessment of ciliary ultrastructure in the diagnosis of PCD over a 20-year period was reviewed. Results During this period, 1182 patients were referred for ciliary ultrastructural analysis, 242 (20%) of whom were confirmed as having the disease. The two main causes of PCD identified were a lack of outer dynein arms (43%) and a lack of both inner and outer dynein arms (24%). Other causes included transposition, radial spoke and inner dynein arm defects. No specific ultrastructural defects were detected in 33 patients (3%) diagnosed as having PCD on the basis of their clinical features and screening tests that included a low nasal nitric oxide concentration or slow saccharine clearance and abnormal ciliary beat frequency or pattern. Conclusions Electron microscopy analysis can confirm but does not always exclude a diagnosis of PCD.

Bronchial mucosal dendritic cells in smokers and ex-smokers with COPD: an electron microscopic study

Background: Bronchial mucosal dendritic cells (DCs) initiate and regulate immune responses to inhaled antigens, viruses and bacteria. Currently, little is known of their numbers in patients with chronic obstructive pulmonary disease (COPD). While reductions in their numbers have been reported recently in smokers with asthma, nothing is known of the effects of cigarette smoking on bronchial DCs in COPD. The present study compares DC numbers in smokers and ex-smokers with COPD. Methods: Endobronchial biopsies were obtained from 15 patients with moderate to severe COPD (10 current smokers with median forced expiratory volume in 1 s (FEV1) 45.5% predicted (range 23–68) and 5 ex-smokers with median FEV1 30% predicted (range 21–52)), 11 non-smokers with asthma (median FEV1 102% predicted (range 89–116)) and 11 non-smoker healthy controls (median FEV1 110% predicted (range 92–135)). Transmission electron microscopy (TEM) was used to identify the total population of DCs by their ultrastructure and their number in the epithelium and subepithelium was counted. Results: Median (range) DC numbers were significantly lower in current smokers with COPD in the epithelium (0.0 (0.0–156.8) cells/mm2) and the subepithelium (4.5 (0.0–63.6) cells/mm2) compared with ex-smokers with COPD (97.9 (93.5–170.3) cells/mm2 in the epithelium (p<0.05); 91.8 (38.2–283.3) cells/mm2 in the subepithelium (p<0.01)). DC numbers in ex-smokers with COPD were similar to those in subjects with atopic asthma and healthy controls (131.6 (33.3–235.5) cells/mm2 in the epithelium and 64.4 (0.0–182.4) cells/mm2 in the subepithelium for the latter). Conclusions: In COPD, bronchial mucosal DC numbers are lower in current smokers while, in those who quit, numbers are similar to non-smoking subjects with asthma and non-smoking healthy controls. The functional consequences of the reduction in mucosal DC numbers in smokers with COPD have yet to be determined.

One very rare and one new tracheal tumour found by electron microscopy: glomus tumour and acinic cell tumour resembling carcinoid tumours by light microscopy.

Tracheal tumours were removed surgically from two patients and diagnosed as carcinoid tumours by routine light microscopy. At a later date, electron microscopy was performed on stored tumour tissue and no neurosecretory granules were found in either case. One showed features of a glomus tumour and the other of an acinic cell tumour. Only two glomus tumours appear to have been reported previously in the trachea, and no acinic cell tumours. Electron microscopy is thus sometimes of great assistance in diagnosing accurately unusual tumours of the lower respiratory tract.

Identification of inorganic dust particles in bronchoalveolar lavage macrophages by energy dispersive x-ray microanalysis.

This study shows that energy dispersive x-ray microprobe analysis to identify and quantify intracellular particles in macrophages obtained by the minimally invasive method of bronchoalveolar lavage (BAL) can detect inorganic dust exposures of many different kinds. Bronchoalveolar lavage macrophages from 22 patients have been examined. Twelve patients had occupational exposure to asbestos, talc, silica, hard metal or printing ink, while 10 had no known history of dust exposure. X-ray microprobe analysis identified particles which related to the known exposures, superimposed on a background of other particles related to smoking (kaolinite and mica) or to the general environment (silicon, titanium, and iron). The particle identification provided useful objective confirmation of the known exposures, except for silica, which could not be distinguished from the general background levels. X-ray microanalysis using BAL macrophages can be helpful for clarification of mixed dust exposures, to identify particles when light microscopy indicates retained dust in patients with no known history of exposure, and to monitor retained particles after removal from exposure.

Role of the cystic fibrosis transmembrane conductance regulator in internalization of Pseudomonas aeruginosa by polarized respiratory epithelial cells

Pseudomonas aeruginosa is an important human pathogen, producing lung infection in individuals with cystic fibrosis (CF), patients who are ventilated and those who are neutropenic. The respiratory epithelium provides the initial barrier to infection. Pseudomonas aeruginosa can enter epithelial cells, although the mechanism of entry and the role of intracellular organisms in its life cycle are unclear. We devised a model of infection of polarized human respiratory epithelial cells with P. aeruginosa and investigated the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in adherence, uptake and IL‐8 production by human respiratory epithelial cells. We found that a number of P. aeruginosa strains could invade and replicate within cells derived from a patient with CF. Intracellular bacteria did not produce host cell cytotoxicity over a period of 24 h. When these cells were transfected with wild‐type CFTR, uptake of bacteria was significantly reduced and release of IL‐8 following infection enhanced. We propose that internalized P. aeruginosa may play an important role in the pathogenesis of infection and that, by allowing greater internalization into epithelial cells, mutant CFTR results in an increased susceptibility of bronchial infection with this microbe.

Development of acute lung injury after the combination of intravenous bleomycin and exposure to hyperoxia in rats.

Pulmonary toxicity is an important adverse effect of bleomycin treatment. Very little is known of the mechanisms underlying the development of lung injury, especially after intravenous administration, or how it can be modulated. In this study acute lung injury induced by bleomycin has been examined in rats by assessment of alveolar lavage cell profiles, histological examination, and measurement of the total pulmonary extravascular albumin space. Intratracheal instillation of bleomycin 1.5 mg resulted in a severe pneumonitis with influx of inflammatory cells into the alveoli as assessed by alveolar lavage, oedema of the alveolar walls, and up to an eight fold increase in the total pulmonary extravascular albumin space, maximal at 72 hours. Intravenous bleomycin 0.15-5 mg produced no detectable injury when assessed in these ways. Exposure to hyperoxia (40-90%) after intravenous bleomycin, however, induced lung injury similar to that produced by intratracheal bleomycin. A much more severe injury followed administration of intravenous bleomycin after an exposure to hyperoxia, which itself resulted in lung injury; but lung injury was still detectable after bleomycin when the exposure to hyperoxia was insufficient to induce changes in control animals. Lung injury was not observed when the exposure to hyperoxia preceded bleomycin treatment. These results indicate the importance of oxygen in the pathways leading to acute lung injury following intravenous bleomycin. We conclude that exposure to oxygen might induce lung injury during and after bleomycin treatment, and suggest that in these circumstances oxygen therapy should be kept to a minimum.

Effect of platelet activating factor on formation and composition of airway fluid in the guinea‐pig trachea.

1. We studied the effect of platelet activating factor (PAF) on leakage of albumin, and secretion of fucose (a marker for mucus glycoprotein) and protein into the tracheal lumen of the guinea‐pig isolated in situ, and on bioelectric properties and fluxes of mannitol in vitro. We also studied the effect of PAF on mucus secretion in human bronchi in vitro. 2. In guinea‐pig, intravenous PAF markedly increased the luminal concentration of protein but did not significantly increase fucose concentrations. Increased albumin leakage (274% above controls at a dose of 50 ng/kg PAF) was associated with the increased luminal content of protein (248% above controls at the same dose of PAF). 3. Leakage of albumin was maximal 10 min after PAF, was significantly reduced by 20 min and had returned to baseline by 30 min. This pattern of leakage could be repeated with successive administrations of PAF. 4. PAF induced small but significant biphasic changes in bioelectric properties in vitro. The initial response was rapid in onset and characterized by maximal increases in short‐circuit current (Isc) of 6.5% above controls at 7.5 min and in conductance (G) of 7% at 20 min. Both responses were blocked by the PAF receptor antagonist WEB 2086. Amiloride blocked the increase in Isc. Permeability of the tissue to mannitol (Pmann) was unaltered. The delayed response was characterized by maximal increases in Isc and G of 10% above controls at 60‐90 min which were not significantly affected by WEB 2086 or amiloride. Pmann was increased by 38% at 90 min. 5. PAF increased fucose secretion in human bronchi in vitro. 6. Lyso‐PAF in vitro caused changes similar to those induced by PAF on bioelectric properties and mucus secretion, but had no significant effects in vivo. 7. Light microscopy showed no evidence of epithelial disruption in animals given intravenous PAF at a dose causing significant albumin transudation. 8. We conclude that PAF increases the protein content of guinea‐pig tracheal fluid principally by inducing plasma leakage rather than mucus secretion and that the small changes in ion transport and epithelial conductance may reduce the tendency to epithelial disruption during plasma leakage.

Pulmonary surfactant composition early in development of acute lung injury after cardiopulmonary bypass: prophylactic use of surfactant therapy.

Cardiopulmonary bypass surgery (CPB) causes lung injury and at least 2% of adult patients and more children develop the most severe form acute respiratory distress syndrome (ARDS). Pulmonary surfactant deficiency contributes to the pathogenesis of ARDS. It has been proposed that surfactant therapy immediately after CPB might arrest progression to ARDS. However, many patients develop only mild lung injury after CPB. Thus early markers are needed to identify those patients at highest risk to guide selection for treatment. The aim of this study was to determine whether changes in surfactant phospholipids occur, and reflect severity of lung injury within the first few hours after bypass. Because of the relatively low incidence of ARDS in adult patients, this study was conducted using young pigs highly susceptible to bypass‐induced lung injury. Eight pigs were given 2 hours bypass. Six controls underwent ‘sham’ bypass. At 3 h after bypass pulmonary vascular endothelial permeability was assessed by transcapillary leakage of radiolabelled transferrin. A 4 hour broncho‐alveolar lavage (BAL) was used to assess intra‐alveolar levels of surfactant, inflammatory cells and oedema protein. Bypass caused falls in arterial oxygenation and lung compliance (P < 0.01), but at this early stage in progression of lung injury BAL surfactant phospholipid and albumin levels were within the control range indicating that the alveolar epithelium had not yet suffered major damage.The main abnormalities were increases in vascular endothelial permeability (P < 0.01), BAL neutrophils (P < 0.01), total protein and sphingomyelin (SM) (P < 0.05). Lung histology showed that the main damage was interstitial oedema located around the bronchioles and their associated vessels. A single instilled dose of surfactant phospholipids in 5 animals caused excess in vivo supplementation and did not reduce the early pathophysiologic changes. Our findings suggest that surfactant phospholipid deficiency does not make a major contribution in the initial stages of lung injury after CPB, and that excessive phospholipid supplementation at this stage can be deleterious.