Hans Lennart Persson

Lysosomal iron in pulmonary alveolar proteinosis : a case report

Pulmonary alveolar proteinosis is characterised by accumulation of surfactant-like material in the distal air spaces. Since lysosomes play a crucial role for degradation of large biomolecules taken up from the cells environment, it was hypothesised that oxidant-induced lysosomal disruption and ensuing cell death might play a role in disease development. In the present study, alveolar macrophages, harvested by whole-lung lavage from a patient diagnosed with pulmonary alveolar proteinosis, are shown to contain large amounts of undigested material within lysosomes, and the same organelle exhibits increased amounts of haemosiderin-bound iron. Compared with murine macrophage-like J774 cells (iron exposed or not), the status of human macrophages was pro-oxidative, i.e. macrophages exhibited a low level of the antioxidant glutathione and large amounts of iron available for Fenton-type chemistry. As a consequence, macrophageal lysosomes were particularly fragile when exposed to physiological concentrations of hydrogen peroxide (generated by glucose oxidase in culture medium). Such lysosomal disruption resulted in extensive cell death by both necrosis and apoptosis independent of caspase-3 activation. Considering the potential role of iron-catalysed oxidant-induced lysosomal rupture and ensuing cell killing for pulmonary alveolar proteinosis pathology and disease progression, whole-lung lavage might be considered early in those cases in which cytochemical staining reveals great numbers of haemosiderin-laden alveolar macrophages.

Vitamin D enhances IL-1β secretion and restricts growth of Mycobacterium tuberculosis in macrophages from TB patients.

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB), the bacterium responsible for tuberculosis (TB), has rekindled the interest in the role of nutritional supplementation of micronutrients, such as vitamin D, as adjuvant treatment. Here, the growth of virulent MTB in macrophages obtained from the peripheral blood of patients with and without TB was studied. The H37Rv strain genetically modified to express Vibrio harveyi luciferase was used to determine the growth of MTB by luminometry in the human monocyte-derived macrophages (hMDMs) from study subjects. Determination of cytokine levels in culture supernatants was performed using a flow cytometry-based bead array technique. No differences in intracellular growth of MTB were observed between the different study groups. However, stimulation with 100nM 1,25-dihydroxyvitamin D significantly enhanced the capacity of hMDMs isolated from TB patients to control the infection. This effect was not observed in hMDMs from the other groups. The interleukin (IL)-1β and IL-10 release by hMDMs was clearly increased upon stimulation with 1,25-dihydroxyvitamin D. Furthermore, the 1,25-dihydroxyvitamin D stimulation also led to elevated levels of TNF-α (tumor necrosis factor-alpha) and IL-12p40. It was concluded that vitamin D triggers an inflammatory response in human macrophages with enhanced secretion of cytokines, as well as enhancing the capacity of hMDMs from patients with active TB to restrict mycobacterial growth.

Quantitative lung SPECT applied on simulated early COPD and humans with advanced COPD

BackgroundReduced ventilation in lung regions affected by chronic obstructive pulmonary disease (COPD), reflected as inhomogeneities in the single-photon emission computed tomography (SPECT) lung image, is correlated to disease advancement. An analysis method for measuring these inhomogeneities is proposed in this work. The first aim was to develop a quantitative analysis method that could discriminate between Monte Carlo simulated normal and COPD lung SPECT images. A second aim was to evaluate the ability of the present method to discriminate between human subjects with advanced COPD and healthy volunteers.MethodsIn the simulated COPD study, different activity distributions in the lungs were created to mimic the healthy lung (normal) and different levels of COPD. Gamma camera projections were Monte Carlo simulated, representing clinically acquired projections of a patient who had inhaled 125 MBq 99mTc-Technegas followed by a 10-min SPECT examination. Reconstructions were made with iterative ordered subset expectation maximisation. The coefficient of variance (CV) was calculated for small overlapping volumes covering the 3D reconstructed activity distribution. A CV threshold value (CVT) was calculated as the modal value of the CV distribution of the simulated normal. The area under the distribution curve (AUC), for CV values greater than CVT, AUC(CVT), was then calculated. Moreover, five patients with advanced emphysema and five healthy volunteers inhaled approximately 75 MBq 99mTc-Technegas immediately before the 20-min SPECT acquisition. In the human study, CVT was based on the mean CV distribution of the five healthy volunteers.ResultsA significant difference (p < 0.001) was found between the Monte-Carlo simulated normal and COPD lung SPECT examinations. The present method identified a total reduction of ventilation of approximately 5%, not visible to the human eye in the reconstructed image. In humans the same method clearly discriminated between the five healthy volunteers and five patients with advanced COPD (p < 0.05).ConclusionsWhile our results are promising, the potential of the AUC(CVT) method to detect less advanced COPD in patients needs further clinical studies.

Leaky lysosomes in lung transplant macrophages: azithromycin prevents oxidative damage

BackgroundLung allografts contain large amounts of iron (Fe), which inside lung macrophages may promote oxidative lysosomal membrane permeabilization (LMP), cell death and inflammation. The macrolide antibiotic azithromycin (AZM) accumulates 1000-fold inside the acidic lysosomes and may interfere with the lysosomal pool of Fe.ObjectiveOxidative lysosomal leakage was assessed in lung macrophages from lung transplant recipients without or with AZM treatment and from healthy subjects. The efficiency of AZM to protect lysosomes and cells against oxidants was further assessed employing murine J774 macrophages.MethodsMacrophages harvested from 8 transplant recipients (5 without and 3 with ongoing AZM treatment) and 7 healthy subjects, and J774 cells pre-treated with AZM, a high-molecular-weight derivative of the Fe chelator desferrioxamine or ammonium chloride were oxidatively stressed. LMP, cell death, Fe, reduced glutathione (GSH) and H-ferritin were assessed.ResultsOxidant challenged macrophages from transplants recipients without AZM exhibited significantly more LMP and cell death than macrophages from healthy subjects. Those macrophages contained significantly more Fe, while GSH and H-ferritin did not differ significantly. Although macrophages from transplant recipients treated with AZM contained both significantly more Fe and less GSH, which would sensitize cells to oxidants, these macrophages resisted oxidant challenge well. The preventive effect of AZM on oxidative LMP and J774 cell death was 60 to 300 times greater than the other drugs tested.ConclusionsAZM makes lung transplant macrophages and their lysososomes more resistant to oxidant challenge. Possibly, prevention of obliterative bronchiolitis in lung transplants by AZM is partly due to this action.

Alveolar macrophages from patients with tuberculosis exhibit reduced capacity of restricting growth of Mycobacterium tuberculosis : a pilot study of vitamin D stimulation in vitro

Background: The role of vitamin D supplementation as adjuvant treatment of tuberculosis (TB) has lately attracted increasing interest. Our aim was to investigate the capacity of alveolar macrophage ...

Oxidant-induced autophagy and ferritin degradation contribute to epithelial–mesenchymal transition through lysosomal iron

Purpose Transforming growth factor (TGF)-β1 triggers epithelial–mesenchymal transition (EMT) through autophagy, which is partly driven by reactive oxygen species (ROS). The aim of this study was to determine whether leaking lysosomes and enhanced degradation of H-ferritin could be involved in EMT and whether it could be possible to prevent EMT by iron chelation targeting of the lysosome. Materials and methods EMT, H-ferritin, and autophagy were evaluated in TGF-β1-stimulated A549 human lung epithelial cells cultured in vitro using Western blotting, with the additional morphological assessment of EMT. By using immunofluorescence and flow cytometry, lysosomes and ROS were assessed by acridine orange and 6-carboxy-2′,7′-dichlorodihydrofluorescein acetate assays, respectively. Results TGF-β1-stimulated cells demonstrated a loss of H-ferritin, which was prevented by the antioxidant N-acetyl-L-cysteine (NAC) and inhibitors of lysosomal degradation. TGF-β1 stimulation generated ROS and autophagosome formation and led to EMT, which was further promoted by the additional ROS-generating cytokine, tumor necrosis factor-α. Lysosomes of TGF-β1-stimulated cells were sensitized to oxidants but also completely protected by lysosomal loading with dextran-bound deferoxamine (DFO). Autophagy and EMT were prevented by NAC, DFO, and inhibitors of autophagy and lysosomal degradation. Conclusion The findings of this study support the role of enhanced autophagic degradation of H-ferritin as a mechanism for increasing the vulnerability of lysosomes to iron-driven oxidant injury that triggers further autophagy during EMT. This study proposes that lysosomal leakage is a novel pathway of TGF-β1-induced EMT that may be prevented by iron-chelating drugs that target the lysosome.

Measures of bronchodilator response of FEV 1 , FVC and SVC in a Swedish general population sample aged 50–64 years, the SCAPIS Pilot Study

Background Data are lacking from general population studies on how to define changes in lung function after bronchodilation. This study aimed to analyze different measures of bronchodilator response of forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC) and slow vital capacity (SVC). Materials and methods Data were derived from the Swedish Cardiopulmonary Bioimage Study (SCAPIS) Pilot study. This analysis comprised 1,050 participants aged 50–64 years from the general population. Participants were investigated using a questionnaire, and FEV1, FVC and SVC were recorded before and 15 minutes after inhalation of 400 µg of salbutamol. A bronchodilator response was defined as the relative change from baseline value expressed as the difference in units of percent predicted normal. Predictors of bronchodilator responses were assessed using multiple linear regression models. Airway obstruction was defined as FEV1/FVC ratio below lower limit of normal (LLN) before bronchodilation, and COPD was defined as an FEV1/FVC ratio below LLN after bronchodilation. Physician-diagnosed asthma was defined as an affirmative answer to “Have you ever had asthma diagnosed by a physician?”. Asymptomatic never-smokers were defined as those not reporting physician-diagnosed asthma, physician-diagnosed COPD or emphysema, current wheeze or chronic bronchitis and being a lifelong never-smoker. Results Among all subjects, the greatest bronchodilator responses (FEV1, FVC and SVC) were found in subjects with asthma or COPD. The upper 95th percentile of bronchodilator responses in asymptomatic never-smokers was 8.7% for FEV1, 4.2% for FVC and 5.0% for SVC. The bronchodilator responses were similar between men and women. In a multiple linear regression model comprising all asymptomatic never-smokers, the bronchodilator response of FEV1 was significantly associated with airway obstruction and height. Conclusion When the bronchodilator response in asymptomatic never-smokers is reported as the difference in units of predicted normal, significant reversibility of FEV1, FVC and SVC to bronchodilators is ~9%, 4% and 5%, respectively.

A retrospective chart review of pirfenidone-treated patients in Sweden: the REPRIS study

Background Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease that usually results in respiratory failure and death. Pirfenidone was approved as the first licensed therapy for IPF in Europe based on phase III trials where patients with a forced vital capacity (FVC) >50% of predicted were included. The aim of this study was to characterise patients treated with pirfenidone in Swedish clinical practice and to describe the adherence to the reimbursement restriction since reimbursement was only applied for patients with FVC below 80% of predicted. Methods This was a retrospective, observational chart review of IPF patients treated with pirfenidone from three Swedish university clinics. Patients initiated on treatment during the period 28 June 2012 to 20 November 2014 were included. Data on patient characteristics, basis of diagnosis, treatment duration, quality of life, and adverse drug reactions (ADRs) were collected from medical charts. Results Forty-four patients were screened and 33 were included in the study. The mean treatment duration from start of pirfenidone until discontinuation or end of study was 38 weeks. At the initiation of pirfenidone treatment, FVC was 62.7% (12.1) [mean (SD)], diffusion capacity (DLco) was 45.1% (13.8) of predicted, and the ratio of forced expiratory volume on 1 sec (FEV1) to FVC was 0.78 (0.1). The percentage of patients with an FVC between 50 and 80% was 87%. Ten of the patients had ADRs including gastrointestinal and skin-related events, cough and signs of impaired hepatic function, but this led to treatment discontinuation in only two patients. Conclusion Data from this chart review showed that adherence to the Swedish reimbursement restriction was followed in the majority of patients during the study period. At the start of pirfenidone treatment, lung function, measured as FVC, was lower in the present cohort of Swedish IPF patients compared with other registry and real-life data. About a third of the patients had ADRs, but discontinuation of the treatment because of ADRs was relatively uncommon.

Increased Lysosomal Membrane Permeabilization in Oxidant-exposed Macrophages of Human Fibrotic Lungs

A disrupted balance of reduced glutathione (GSH) and iron (Fe) and subsequent enhanced susceptibility of lysosomes of lung macrophages (LMs) to oxidants may play a role in lung fibrogenesis. We assessed cellular Fe/GSH, lysosomal membrane permeabilization (LMP), and cell death in cultures of oxidant exposed LMs. LMs from 7 lung fibrosis patients and healthy subjects were exposed to a physiologic concentration of H2O2 for 1 h. LMP was assessed with acridine orange green fluorescence, apoptosis/necrosis were estimated by apoptotic DNA and typical morphology, Fe was assessed with Prussian blue staining/atomic absorption spectrophotometry, and GSH was evaluated using a GSH assay kit. Oxidant-induced LMP and cell death were more pronounced in cultures of LMs from patients with lung fibrosis, and these cells contained less GSH and more cytochemically stained Fe. These observations indicate that LMP may be involved in fibrosis development, possibly through activation of the inflammasome complex. Further studies are warranted for a detailed understanding.

The influence of disease severity and lifestyle factors on the peak annual 25(OH)D value of COPD patients

Background The prevalence of individuals deficient in vitamin D (defined as a serum level of the stable metabolite 25(OH)D <50 nmol/L) is increasing in countries with low annual ultraviolet (UV) radiation and among individuals unable to perform outdoor activities, for example, COPD patients. Objective To assess the role of vitamin D deficiency, independently of seasonal variation, the peak annual value of 25(OH)D was measured in subjects with advanced COPD ± long-term oxygen therapy (LTOT) and lung healthy control subjects. A method to grade the individual annual UV light exposure was designed and tested. Subjects and methods Sixty-six Caucasians with advanced COPD (28 with LTOT) and 47 control subjects were included, and the levels of 25(OH)D were determined in late summer/early fall when the annual peak was assumed. Questionnaires about COPD symptoms, general health, lifestyle, dietary habits and QoL were used to collect data. Lung function tests and blood sampling were performed. Results The peak annual 25(OH)D of COPD subjects was significantly lower than in the control subjects, but there was no significant difference between COPD patients with and without LTOT. Ongoing vitamin D supplementation was the single most important intervention to maintain 25(OH)D levels ≥50 nmol/L. Among vitamin D-deficient COPD subjects, 25(OH) D correlated positively with forced expiratory volume in 1 second as % predicted, Modified British Medical Research Council score, blood oxygenation, food portion size, Mediterranean Diet Score and Ultraviolet Score. Conclusion Vitamin D deficiency was common among healthy individuals and COPD subjects. Peak annual 25(OH)D levels of COPD subjects correlated with clinically important outcomes. The present study emphasizes the need to routinely monitor vitamin D status among patients with advanced COPD and to consider to medicate those with vitamin D deficiency with vitamin D supplementation.