4-Phenylbutyric acid treatment rescues trafficking and processing of a mutant surfactant protein C

Mutations in the SFTPC gene, encoding surfactant protein–C (SP-C), are associated with interstitial lung disease (ILD). Knowledge of the intracellular fate of mutant SP-C is essential in the design of therapies to correct trafficking/processing of the proprotein, and to prevent the formation of cytotoxic aggregates. We assessed the potential of a chemical chaperone to correct the trafficking and processing of three disease-associated mutant SP-C proteins. HEK293 cells were stably transfected with wild-type (SP-C(WT)) or mutant (SP-C(L188Q), SP-C(Δexon4), or SP-C(I73T)) SP-C, and cell lines with a similar expression of SP-C mRNA were identified. The effects of the chemical chaperone 4-phenylbutyric acid (PBA) and lysosomotropic drugs on intracellular trafficking to the endolysosomal pathway and the subsequent conversion of SP-C proprotein to mature peptide were assessed. Despite comparable SP-C mRNA expression, proprotein concentrations varied greatly: SP-C(I73T) was more abundant than SP-C(WT) and was localized to the cell surface, whereas SP-C(Δexon4) was barely detectable. In contrast, SP-C(L188Q) and SP-C(WT) proprotein concentrations were comparable, and a small amount of SP-C(L188Q) was localized to the endolysosomal pathway. PBA treatment restored the trafficking and processing of SP-C(L188Q) to SP-C(WT) concentrations, but did not correct the mistrafficking of SP-C(I73T) or rescue SP-C(Δexon4). PBA treatment also promoted the aggregation of SP-C proproteins, including SP-C(L188Q). This study provides proof of the principle that a chemical chaperone can correct the mistrafficking and processing of a disease-associated mutant SP-C proprotein

Comparative Proteomic Analysis of Lung Lamellar Bodies and Lysosome-Related Organelles

Pulmonary surfactant is a complex mixture of lipids and proteins that is essential for postnatal function. Surfactant is synthesized in alveolar type II cells and stored as multi-bilayer membranes in a specialized secretory lysosome-related organelle (LRO), known as the lamellar body (LB), prior to secretion into the alveolar airspaces. Few LB proteins have been identified and the mechanisms regulating formation and trafficking of this organelle are poorly understood. Lamellar bodies were isolated from rat lungs, separated into limiting membrane and core populations, fractionated by SDS-PAGE and proteins identified by nanoLC-tandem mass spectrometry. In total 562 proteins were identified, significantly extending a previous study that identified 44 proteins in rat lung LB. The lung LB proteome reflects the dynamic interaction of this organelle with the biosynthetic, secretory and endocytic pathways of the type II epithelial cell. Comparison with other LRO proteomes indicated that 60% of LB proteins were detected in one or more of 8 other proteomes, confirming classification of the LB as a LRO. Remarkably the LB shared 37.8% of its proteins with the melanosome but only 9.9% with lamellar bodies from the skin. Of the 229 proteins not detected in other LRO proteomes, a subset of 34 proteins was enriched in lung relative to other tissues. Proteins with lipid-related functions comprised a significant proportion of the LB unique subset, consistent with the major function of this organelle in the organization, storage and secretion of surfactant lipid. The lung LB proteome will facilitate identification of molecular pathways involved in LB biogenesis, surfactant homeostasis and disease pathogenesis

Can an epilepsy nurse specialist-led self-management intervention reduce attendance at emergency departments and promote well-being for people with severe epilepsy?:A non-randomised trial with a nested qualitative phase

Objectives: To (1) describe the characteristics and service use of people with established epilepsy (PWE) who attend the emergency department (ED); (2) evaluate the economic impact of PWE who attend the ED; (3) determine the effectiveness and cost-effectiveness of an epilepsy nurse specialist (ENS)-led self-management intervention plus treatment as usual (TAU) compared with TAU alone in reducing ED use and promoting well-being; (4) describe patients' views of the intervention; and (5) explore their reasons for attending the ED. Design: Non-randomised trial with nested qualitative study. Setting: The EDs of three inner London hospitals. The EDs each offer similar services and support a similar local population, which made a comparison of patient outcomes reasonable. Participants: Adults diagnosed with epilepsy for ≥ 1 year were prospectively identified from the EDs by presenting symptom/discharge diagnosis. We recruited 85 of 315 patients with 44 forming the intervention group and 41 the comparison group. Intervention: Intervention participants were offered two one-to-one outpatient sessions delivered by an ENS who aimed to optimise self-management skills and knowledge of appropriate emergency service use. The first session lasted for 45–60 minutes and the second for 30 minutes. Main outcome measures: The primary outcome was the number of ED visits that participants reported making over the 6 months preceding the 12-month follow-up. Secondary outcomes were visits reported at the 6-month follow-up and scores on psychosocial measures. Results: In the year preceding recruitment, the 85 participants together made 270 ED visits. The frequency of their visits was positively skewed, with 61% having attended multiple times. The mean number of visits per participant was 3.1 [standard deviation (SD) 3.6] and the median was two (interquartile range 1–4). Mean patient service cost was £2355 (SD £2455). Compared with findings in the general epilepsy population, participants experienced more seizures and had greater anxiety, lower epilepsy knowledge and greater perceived stigma. Their outpatient care was, however, consistent with National Institute for Health and Clinical Excellence recommendations. In total, 81% of participants were retained at the 6- and 12-month follow-ups, and 80% of participants offered the intervention attended. Using intention-to-treat analyses, including those adjusted for baseline differences, we found no significant effect of the intervention on ED use at the 6-month follow-up [adjusted incidence rate ratio (IRR) 1.75, 95% confidence interval (CI) 0.93 to 3.28] or the 12-month follow-up (adjusted IRR 1.92, 95% CI 0.68 to 5.41), nor on any psychosocial outcomes. Because they spent less time as inpatients, however, the average service cost of intervention participants over follow-up was less than that of TAU participants (adjusted difference £558, 95% CI –£2409 to £648). Lower confidence in managing epilepsy and more felt stigma at baseline best predicted more ED visits over follow-up. Interviews revealed that patients generally attended because they had no family, friend or colleague nearby who had the confidence to manage a seizure. Most participants receiving the intervention valued it, including being given information on epilepsy and an opportunity to talk about their feelings. Those reporting most ED use at baseline perceived the most benefit. Conclusions: At baseline, > 60% of participants who had attended an ED in the previous year had reattended in the same year. In total, 50% of their health service costs were accounted for by ED use and admissions. Low confidence in their ability to manage their epilepsy and a greater sense of stigma predicted frequent attendance. The intervention did not lead to a reduction in ED use but did not cost more, partly because those receiving the intervention had shorter average hospital stays. The most common reason reported by PWE for attending an ED was the lack of someone nearby with sufficient experience of managing a seizure. Those who attended an ED frequently and received the intervention were more likely to report that the intervention helped them. Our findings on predictors of ED use clarify what causes ED use and suggest that future interventions might focus more on patients' perceptions of stigma and on their confidence in managing epilepsy. If addressed, ED visits might be reduced and efficiency savings generated. Trial registration: Current Controlled Trials ISRCTN06469947. Funding: This project was funded by the NIHR Health Services and Delivery Research programme and will be published in full in Health Services and Delivery Research; Vol. 1, No. 9. See the HSDR programme website for further project information

Overexpression of lunatic fringe does not affect epithelial cell differentiation in the developing mouse lung

The Notch/Notch-ligand pathway regulates cell fate decisions and patterning in various tissues. Several of its components are expressed in the developing lung, suggesting that this pathway is important for airway cellular patterning. Fringe proteins, which modulate Notch signaling, are crucial for defining morphogenic borders in several organs. Their role in controlling cellular differentiation along anterior-posterior axis of the airways is unknown. Herein, we report the temporal-spatial expression patterns of Lunatic fringe (Lfng) and Notch-regulated basic helix-loop-helix factors, Hes1 and Mash-1, during murine lung development. Lfng was only expressed during early development in epithelial cells lining the larger airways. Those epithelial cells also expressed Hes1, but at later gestation Hes1 expression was confined to epithelium lining the terminal bronchioles. Mash-1 displayed a very characteristic expression pattern. It followed neural crest migration in the early lung, whereas at later stages Mash-1 was expressed in lung neuroendocrine cells. To clarify whether Lfng influences airway cell differentiation, Lfng was overexpressed in distal epithelial cells of the developing mouse lung. Overexpression of Lfng did not affect spatial or temporal expression of Hes1 and Mash-1. Neuroendocrine CGRP and protein gene product 9.5 expression was not altered by Lfng overexpression. Expression of proximal ciliated (β-tubulin IV), nonciliated (CCSP), and distal epithelial cell (SP-C, T1α) markers also was not influenced by Lfng excess. Overexpression of Lfng had no effect on mesenchymal cell marker (α-sma, vWF, PECAM-1) expression. Collectively, the data suggest that Lunatic fringe does not play a significant role in determining cell fate in fetal airway epithelium

Iroquois genes influence proximo-distal morphogenesis during rat lung development

textabstractLung development is a highly regulated process directed by mesenchymal-epithelial interactions, which coordinate the temporal and spatial expression of multiple regulatory factors required for proper lung formation. The Iroquois homeobox (Irx) genes have been implicated in the patterning and specification of several Drosophila and vertebrate organs, including the heart. Herein, we investigated whether the Irx genes play a role in lung morphogenesis. We found that Irx1-3 and Irx5 expression was confined to the branching lung epithelium, whereas Irx4 was not expressed in the developing lung. Antisense knockdown of all pulmonary Irx genes together dramatically decreased distal branching morphogenesis and increased distention of the proximal tubules in vitro, which was accompanied by a reduction in surfactant protein C-positive epithelial cells and an increase in β-tubulin IV and Clara cell secretory protein positive epithelial structures. Transmission electron microscopy confirmed the proximal phenotype of the epithelial structures. Furthermore, antisense Irx knockdown resulted in loss of lung mesenchyme and abnormal smooth muscle cell formation. Expression of fibroblast growth factors (FGF) 1, 7, and 10, FGF receptor 2, bone morphogenetic protein 4, and Sonic hedgehog (Shh) were not altered in lung explants treated with antisense Irx oligonucleotides. All four Irx genes were expressed in Shh- and Gli 2-deficient murine lungs. Collectively, these results suggest that Irx genes are involved in the regulation of proximo-distal morphogenesis of the developing lung but are likely not linked to the FGF, BMP, or Shh signaling pathways. Copyrigh