Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.     

An improved formulation of the zirconyl hematoxylin stain for acidic mucins

Zirconyl hematoxylin stains acidic mucins darkly and specifically using a solution of 100 mg hematoxylin, 5 ml ethanol, 5 ml 0.5% sodium iodate, 400 mg zirconyl chloride octahydrate, and 30 ml 25% aqueous glycerol. The stain is especially advantageous for studying goblet cells and Paget cells.     

Low-dose CT measurements of airway dimensions and emphysema associated with airflow limitation in heavy smokers: a cross sectional study

Background

Increased airway wall thickness (AWT) and parenchymal lung destruction both contribute to airflow limitation. Advances in computed tomography (CT) post-processing imaging allow to quantify these features. The aim of this Dutch population study is to assess the relationships between AWT, lung function, emphysema and respiratory symptoms.

Methods

AWT and emphysema were assessed by low-dose CT in 500 male heavy smokers, randomly selected from a lung cancer screening population. AWT was measured in each lung lobe in cross-sectionally reformatted images with an automated imaging program at locations with an internal diameter of 3.5 mm, and validated in smaller cohorts of patients. The 15^th percentile method (Perc15) was used to assess the severity of emphysema. Information about respiratory symptoms and smoking behavior was collected by questionnaires and lung function by spirometry.

Results

Median AWT in airways with an internal diameter of 3.5 mm (AWT_3.5) was 0.57 (0.44 - 0.74) mm. Median AWT in subjects without symptoms was 0.52 (0.41-0.66) and in those with dyspnea and/or wheezing 0.65 (0.52-0.81) mm (p<0.001). In the multivariate analysis only AWT_3.5 and emphysema independently explained 31.1%and 9.5%of the variance in FEV₁%predicted, respectively, after adjustment for smoking behavior.

Conclusions

Post processing standardization of airway wall measurements provides a reliable and useful method to assess airway wall thickness. Increased airway wall thickness contributes more to airflow limitation than emphysema in a smoking male population even after adjustment for smoking behavior.     

Prolonged cigarette smoke exposure alters mitochondrial structure and function in airway epithelial cells

Background

Cigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis.

Methods

We studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls.

Results

We observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1.

Conclusion

The observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD.     

What does biologically meaningful mean? A perspective on gene regulatory network validation

Gene regulatory networks (GRNs) are rapidly being delineated, but their quality and biological meaning are often questioned. Here, I argue that biological meaning is challenging to define and discuss reasons why GRN validation should be interpreted cautiously.     

Predicting expected progeny difference for marbling score in Angus cattle using artificial neural networks and Bayesian regression models

Background

Artificial neural networks (ANN) mimic the function of the human brain and are capable of performing massively parallel computations for data processing and knowledge representation. ANN can capture nonlinear relationships between predictors and responses and can adaptively learn complex functional forms, in particular, for situations where conventional regression models are ineffective. In a previous study, ANN with Bayesian regularization outperformed a benchmark linear model when predicting milk yield in dairy cattle or grain yield of wheat. Although breeding values rely on the assumption of additive inheritance, the predictive capabilities of ANN are of interest from the perspective of their potential to increase the accuracy of prediction of molecular breeding values used for genomic selection. This motivated the present study, in which the aim was to investigate the accuracy of ANN when predicting the expected progeny difference (EPD) of marbling score in Angus cattle. Various ANN architectures were explored, which involved two training algorithms, two types of activation functions, and from 1 to 4 neurons in hidden layers. For comparison, BayesCπ models were used to select a subset of optimal markers (referred to as feature selection), under the assumption of additive inheritance, and then the marker effects were estimated using BayesCπ with π set equal to zero. This procedure is referred to as BayesCpC and was implemented on a high-throughput computing cluster.

Results

The ANN with Bayesian regularization method performed equally well for prediction of EPD as BayesCpC, based on prediction accuracy and sum of squared errors. With the 3K-SNP panel, for example, prediction accuracy was 0.776 using BayesCpC, and ranged from 0.776 to 0.807 using BRANN. With the selected 700-SNP panel, prediction accuracy was 0.863 for BayesCpC and ranged from 0.842 to 0.858 for BRANN. However, prediction accuracy for the ANN with scaled conjugate gradient back-propagation was lower, ranging from 0.653 to 0.689 with the 3K-SNP panel, and from 0.743 to 0.793 with the selected 700-SNP panel.

Conclusions

ANN with Bayesian regularization performed as well as linear Bayesian regression models in predicting additive genetic values, supporting the idea that ANN are useful as universal approximators of functions of interest in breeding contexts.     

Microtubules and cyclic amp in human leukocytes: on the order of things

We have shown previously that the β-adrenergic agonist isoproterenol (2μM) and the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) produce a much greater increase in cyclic AMP in human leukocytes that have been pretreated with colchicine (or with other agents that affect microtubule assembly) than in control leukocytes. The effects of colchicines were both time- and dose-dependant. These and other data suggested that the generation of cyclic AMP is normally restricted by an intact system of cytoplasmic microtubules. If so, then the same time and dose dependencies might apply to other colchicines-induced changes in leukocyte function. We have now assayed the distribution of concanavalin A (Con A)-receptor complexes on the leukocyte membrane, taking into account that leukocytes competent to assemble microtubules show a uniform distribution of surface- bound Con A whereas microtubule-deficient cells accumulate Con A in surface caps. We have found that the effect of colchicine on capping is also both time- and dose dependent, and that the dose-response relationships conform to those required to increase cyclic AMP levels. These findings provide further evidence that both colchicine-induced Con-A capping and colchicine- induced cyclic AMP generation depend upon the relaxation of constraints normally imposed by cytoplasmic microtubules upon the plasma membrane, which limit, respectively, lateral mobility of the lectin-receptor complexes, and expression of hormone-sensitive adenylate cyclase. Moreover, colchicine-induced Con-A cap formation is not affected even by very large changes in leukocyte cyclic AMP levels. Thus, elevated cyclic AMP levels do not appear to promote the dissolution of microtubules; rather, the dissolution of microtubules permits the generation of increased amounts of cyclic AMP.     

Multidisciplinary transmural rehabilitation for older persons with a stroke: the design of a randomised controlled trial

Background

Stroke is one of the major causes of loss of independence, decreased quality of life and mortality among elderly people. About half of the elderly stroke patients discharged after rehabilitation in a nursing home still experience serious impairments in daily functioning one year post stroke, which can lead to difficulties in picking up and managing their social life. The aim of this study is to evaluate the effectiveness and feasibility of a new multidisciplinary transmural rehabilitation programme for older stroke patients.

Methods

A two group multicentre randomised controlled trial is used to evaluate the effects of the rehabilitation programme. The programme consists of three care modules: 1) neurorehabilitation treatment for elderly stroke patients; 2) empowerment training for patient and informal caregiver; and 3) stroke education for patient and informal caregiver. The total programme has a duration of between two and six months, depending on the individual problems of the patient and informal caregiver. The control group receives usual care in the nursing home and after discharge.Patients aged 65 years and over are eligible for study participation when they are admitted to a geriatric rehabilitation unit in a nursing home due to a recent stroke and are expected to be able to return to their original home environment after discharge. Data are gathered by face-to-face interviews, self-administered questionnaires, focus groups and registration forms. Primary outcomes for patients are activity level after stroke, functional dependence, perceived quality of life and social participation. Outcomes for informal caregivers are perceived care burden, objective care burden, quality of life and perceived health. Outcome measures of the process evaluation are implementation fidelity, programme deliverance and the opinion of the stroke professionals, patients and informal caregivers about the programme. Outcome measures of the economic evaluation are the healthcare utilisation and associated costs. Data are collected at baseline, and after six and 12 months. The first results of the study will be expected in 2014.

Trial registration

International Standard Randomised Controlled Trial Register Number ISRCTN62286281, The Dutch Trial Register NTR2412

     

Stimulation of allergen-loaded macrophages by TLR9-ligand potentiates IL-10-mediated suppression of allergic airway inflammation in mice

BackgroundPreviously, we demonstrated that OVA-loaded macrophages (OVA-Mφ) partially suppress OVA-induced airway manifestations of asthma in BALB/c mice. In vitro studies showed that OVA-Mφ start to produce IL-10 upon interaction with allergen-specific T cells, which might mediate their immunosuppressive effects. Herein, we examined whether IL-10 is essential for the immunosuppressive effects of OVA-Mφ in vivo, and whether ex vivo stimulation of the IL-10 production by OVA-Mφ could enhance these effects.MethodsPeritoneal Mφ were loaded with OVA and stimulated with LPS or immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) in vitro. The increase of IL-10 production was examined and, subsequently, ex vivo stimulated OVA-Mφ were used to treat (i.v.) OVA-sensitized mice. To further explore whether Mφ-derived IL-10 mediates the immunosuppressive effects, Mφ isolated from IL-10^-/- mice were used for treatment.ResultsWe found that stimulation with LPS or ISS-ODN highly increased the IL-10 production by OVA-Mφ (2.5-fold and 4.5-fold increase, respectively). ISS-ODN stimulation of OVA-Mφ significantly potentiated the suppressive effects on allergic airway inflammation. Compared to sham-treatment, ISS-ODN-stimulated OVA-Mφ suppressed the airway eosinophilia by 85% (vs. 30% by unstimulated OVA-Mφ), IL-5 levels in bronchoalveolar lavage fluid by 80% (vs. 50%) and serum OVA-specific IgE levels by 60% (vs. 30%). Importantly, IL-10^-/-Mφ that were loaded with OVA and stimulated with ISS-ODN ex vivo, failed to suppress OVA-induced airway inflammation.ConclusionsThese results demonstrate that Mφ-derived IL-10 mediates anti-inflammatory responses in a mouse model of allergic asthma, which both can be potentiated by stimulation with ISS-ODN.