Janine Koepke

Detection of obstructive sleep apnoea by an electronic nose

Diagnosis of obstructive sleep apnoea syndrome (OSAS) is technically demanding, cost-intensive and time-consuming. The measurement of volatile organic compounds by an electronic nose is an innovative method that determines distinct molecular patterns of exhaled breath in different patient groups. We addressed the following questions: What is the diagnostic accuracy of an electronic nose in the detection of OSAS and the ability to detect effects of standard therapy in patients with OSAS? Are these results related to changes in distinct markers of airway inflammation and extracellular remodelling? We included 40 OSAS patients and 20 healthy controls. Exhaled breath of all participants was analysed using the Cyranose 320 electronic nose. Pharyngeal washings were performed to sample the upper airway compartment. For statistical analysis linear discriminant analysis was employed. We identified a linear discriminant function separating OSAS from control (p<0.0001). The corresponding area under the receiver-operating curve was 0.85 (95% CI 0.75–0.96; sensitivity 0.93 and specificity 0.7). In pharyngeal washing fluids of OSAS patients, we observed higher levels of &agr;1-antitrypsin and markers of extracellular remodelling compared to controls. The electronic nose can distinguish between OSAS patients and controls with high accuracy.

Benefits of whole body vibration training in patients hospitalised for COPD exacerbations - a randomized clinical trial

BackgroundPatients with stable COPD show improvements in exercise capacity and muscular function after the application of whole body vibration. We aimed to evaluate whether this modality added to conventional physiotherapy in exacerbated hospitalised COPD patients would be safe and would improve exercise capacity and quality of life.Methods49 hospitalised exacerbated COPD patients were randomized (1:1) to undergo physiotherapy alone or physiotherapy with the addition of whole body vibration. The primary endpoint was the between-group difference of the 6-minute walking test (day of discharge – day of admission). Secondary assessments included chair rising test, quality of life, and serum marker analysis.ResultsWhole body vibration did not cause procedure-related adverse events. Compared to physiotherapy alone, it led to significantly stronger improvements in 6-minute walking test (95.55 ± 76.29 m vs. 6.13 ± 81.65 m; p = 0.007) and St. Georges Respiratory Questionnaire (-6.43 ± 14.25 vs. 5.59 ± 19.15, p = 0.049). Whole body vibration increased the expression of the transcription factor peroxisome proliferator receptor gamma coactivator-1-α and serum levels of irisin, while it decreased serum interleukin-8.ConclusionWhole body vibration during hospitalised exacerbations did not cause procedure-related adverse events and induced clinically significant benefits regarding exercise capacity and health-related quality of life that were associated with increased serum levels of irisin, a marker of muscle activity.Trial registrationGerman Clinical Trials Register DRKS00005979. Registered 17 March 2014.

The effects of weekly augmentation therapy in patients with PiZZ α1-antitrypsin deficiency

Background The major concept behind augmentation therapy with human α1-antitrypsin (AAT) is to raise the levels of AAT in patients with protease inhibitor phenotype ZZ (Glu342Lys)-inherited AAT deficiency and to protect lung tissues from proteolysis and progression of emphysema. Objective To evaluate the short-term effects of augmentation therapy (Prolastin®) on plasma levels of AAT, C-reactive protein, and chemokines/cytokines. Materials and methods Serum and exhaled breath condensate were collected from individuals with protease inhibitor phenotype ZZ AAT deficiency-related emphysema (n = 12) on the first, third, and seventh day after the infusion of intravenous Prolastin. Concentrations of total and polymeric AAT, interleukin-8 (IL-8), monocyte chemotactic protein-1, IL-6, tumor necrosis factor-α, vascular endothelial growth factor, and C-reactive protein were determined. Blood neutrophils and primary epithelial cells were also exposed to Prolastin (1 mg/mL). Results There were significant fluctuations in serum (but not in exhaled breath condensate) levels of AAT polymers, IL-8, monocyte chemotactic protein-1, IL-6, tumor necrosis factor-α, and vascular endothelial growth factor within a week of augmentation therapy. In general, augmented individuals had higher AAT and lower serum levels of IL-8 than nonaugmented subjects. Prolastin added for 3 hours to neutrophils from protease inhibitor phenotype ZZ individuals in vitro reduced IL-8 release but showed no effect on cytokine/chemokine release from human bronchial epithelial cells. Conclusion Within a week, augmentation with Prolastin induced fluctuations in serum levels of AAT polymers and cytokine/chemokines but specifically lowered IL-8 levels. It remains to be determined whether these effects are related to the Prolastin preparation per se or to the therapeutic efficacy of augmentation with AAT.

α1-antitrypsin modulates microglial-mediated neuroinflammation and protects microglial cells from amyloid-β-induced toxicity.

BackgroundOne hallmark of Alzheimer disease is microglial activation. Therapeutic approaches for this neurodegenerative disease include the modulation of microglial cells. α1-antitrypsin (A1AT) has been shown to exert anti-inflammatory effects on macrophages and lung epithelial cells and an inhibition of calpain activity in neutrophil granulocytes. Nothing is known about the effect of A1AT on microglial-mediated neuroinflammation. Our aim was to investigate the effect of A1AT on amyloid-β (Aβ)- and LPS-treated microglial cells in vitro with respect to cytokine production, stress pathways, cell viability, phagocytotic abilities and the underlying mechanisms.MethodsPrimary microglial cells were isolated from Swiss Webster mouse embryos on embryonic day 13.5. Cytokines in the supernatants of treated primary microglial cells were analyzed with ELISAs, and accumulated nitrite was detected with Griess reagents. Intracellular stress pathways were investigated in cell lysates using western blotting. Intracellular calcium levels were detected in BV-2 microglial cells loaded with the Ca2+-sensitive (fluorescent) dye Fluo-4. Calpain activity in primary microglial cells was assessed by using a calpain activity assay. Cell viability of Aβ-treated microglial cells was analyzed using MTT assay. Phagocytosis of Aβ was evaluated with western blot analysis.ResultsUpon co-administration, A1AT reduced pro-inflammatory mediators induced by LPS or Aβ. Interestingly, we detected a reduction in calpain activity and in the concentration of intracellular calcium that might mediate the anti-inflammatory effects of A1AT. Inhibition of the classic activation pathways, such as phosphorylation of mitogen-activated protein kinases or activation of protein kinase A were excluded as a mechanism of A1AT-mediated effects. In addition, A1AT increased the viability of Aβ-treated microglial cells and reduced Aβ phagocytosis.ConclusionsWe provide evidence on the mechanism of action of A1AT on microglial-mediated neuroinflammation in vitro. Our in vitro data indicate that A1AT treatment modulates microglial cells in inflammatory conditions and that this modulation is due to an inhibition of calpain activity and intracellular calcium levels. The underlying mechanisms of the effects observed here are promising for future therapeutic strategies and should thus be further pursued in transgenic mouse models of Alzheimer disease.

Benefits of High-Intensity Exercise Training to Patients with Chronic Obstructive Pulmonary Disease: A Controlled Study

Background: Various exercise training programs are used for patients with chronic obstructive pulmonary disease (COPD) of different severity. Objectives: To investigate the impact of individualized high-intensity training on exercise capacity with COPD. Methods: A total of 49 patients agreed to participate. Of these, 31 were assigned to the training group and 18 served as controls. The training group exercised twice a week for 90 min with consecutively increasing loads. At the time of enrollment (T0), as well as after 3 (T1) and 6 (T2) months, a 6-min walk test (6-MWT) was performed and data on health-related quality of life, femoral muscle thickness, and various serum markers were obtained. Results: The training group improved in their 6-MWT results (T0 = 407 ± 152 m vs. T1 = 459 ± 127 m, p = 0.002, vs. T2 = 483.2 ± 130.1 m, p = 0.004), in their cross-sectional area of the musculus rectus femoris (T0 = 6.2 ± 1.2 cm2 vs. T1 = 6.9 ± 1.2 cm2, p = 0.003, vs. 7.5 ± 1.6 cm2, p = 0.002), and in their St. Georges Respiratory Questionnaire (SGRQ) score (T0 = 43.3 ± 18.0 vs. T1 = 36.0 ± 18.4, p = 0.001, vs. T2 = 34.7 ± 18. 0, p = 0.004). Serum levels of myostatin, irisin, resistin, and α-Klotho did not change significantly within the training period. Of note, the exercise group showed an inverse relationship between serum levels of resistin and those of α-Klotho after 6 months (r = -0.608, p = 0.021). Conclusions: COPD patients undergoing an individualized, structured, high-intensity training program improved their exercise capacity, gained muscle mass, and improved their quality of life.

Does augmentation with alpha1-antitrypsin affect neutrophil extracellular traps formation?

Alpha1-Antitrypsin (AAT) is a major circulating inhibitor of neutrophil proteases, specifically elastase that has broad substrate specificity and can degrade many connective tissue matrix constituents. Severe inherited Z (Glu342Lys) deficiency of AAT (AATD) is characterized by a low serum AAT level (less than 20% of normal which is 11μM/L), the protease/antiprotease balance shift in favor of the elastase and an increased risk of early onset pulmonary emphysema. This lends support to the protease-antiprotease imbalance theory of the pathogenesis of emphysema and to AAT augmentation therapy.

Therapy with plasma purified alpha1-antitrypsin (Prolastin®) induces time-dependent changes in plasma levels of MMP-9 and MPO.

The common Z mutation (Glu342Lys) of α1-antitrypsin (A1AT) results in the polymerization and intracellular retention of A1AT protein. The concomitant deficiency of functional A1AT predisposes PiZZ subjects to early onset emphysema. Clinical studies have implied that, among the biomarkers associated with emphysema, matrix metalloproteinase 9 (MMP-9) is of particular importance. Increased plasma MMP-9 levels are proposed to predict the decline of lung function as well as greater COPD exacerbations in A1AT deficiency-associated emphysema. The aim of the present study was to investigate the effect of A1AT therapy (Prolastin) on plasma MMP-9 and myeloperoxidase (MPO) levels. In total 34 PiZZ emphysema patients were recruited: 12 patients without and 22 with weekly intravenous (60 mg/kg body weight) A1AT therapy. The quantitative analysis of A1AT, MMP-9 and MPO was performed in serum and in supernatants of blood neutrophils isolated from patients before and after therapy. Patients with Prolastin therapy showed significantly lower serum MMP-9 and MPO levels than those without therapy. However, parallel analysis revealed that a rapid infusion of Prolastin is accompanied by a transient elevation of plasma MMP-9 and MPO levels. Experiments with freshly isolated blood neutrophils confirmed that therapy with Prolastin causes transient MMP-9 and MPO release. Prolastin induced the rapid release of MMP-9 and MPO when added directly to neutrophil cultures and this reaction was associated with the presence of IgA in A1AT preparation. Our data support the conclusion that changes in plasma levels of MMP-9 and MPO mirror the effect of Prolastin on blood neutrophils.

Reduction of glutamate-induced excitotoxicity in murine primary neurons involving calpain inhibition

Excessive glutamate secretion leads to excitotoxicity, which has been shown to underlie neurodegenerative disorders. Excitotoxicity is in part exerted by overactivation of calpains, which promote neuronal cell death via induction of limited proteolysis of the cellular proteins p35, regulatory subunit of cyclin-dependent kinase 5, and αII-spectrin. We used primary murine neuronal cells in a model of glutamate toxicity. The protease inhibitor α1-antitrypsin was able to prevent glutamate toxicity as determined by MTT assay and immunofluorescence. Calpain and caspase 3 activity were reduced following α1-antitrypsin treatment, as assessed by calpain and caspase 3 activity assays. In addition we could observe a modulation of cleavage of the calpain/caspase substrates αII-spectrin and p35 in Western blots. In summary, α1-antitrypsin shows inhibitory effects on excitotoxicity of primary neurons involving the inhibition of calpain activity. The advantage of using α1-antitrypsin is that the substance is already in clinical use for the treatment of patients with hereditary α1-antitrypsin deficiency. Further experiments are required in animal models of neurodegenerative disorders to assess the suitability of this substance in patients suffering from Alzheimers disease or Parkinsons disease.

How can we improve the detection of Alpha1-antitrypsin deficiency?

The Z deficiency in α1-antitrypsin (A1ATD) is an under-recognized condition. Alpha1-antitrypsin (A1AT) is the main protein in the α1-globulin fraction of serum protein electrophoresis (SPE); however, evaluation of the α1-globulin protein fraction has received very little attention. Serum Z-type A1AT manifests in polymeric forms, but their interference with quantitative immunoassays has not been reported. Here, 214 894 samples were evaluated by SPE at the G. Fracastoro Hospital of Verona, Italy. Patients with an A1AT level ≤ 0.92 g/L were recalled to complete A1ATD diagnosis. In parallel, to qualitatively and quantitatively characterize A1AT, sera samples from 10 PiZZ and 10 PiMM subjects obtained at the National Institute of Tuberculosis and Lung Diseases in Warsaw, Poland, were subjected to non-denaturing 7.5% PAGE and 7.5% SDS-PAGE followed by Western blot. Moreover, purified A1AT was heated at 60°C and analyzed by a non-denaturing PAGE and 4–15% gradient SDS-PAGE followed by Western blot as well as by isolelectrofocusing and nephelometry. A total of 966 samples manifested percentages ≤ 2.8 or a double band in the alpha1-zone. According to the nephelometry data, 23 samples were classified as severe (A1AT ≤ 0.49 g/L) and 462 as intermediate (A1AT >0.49≤ 1.0 g/L) A1ATD. Twenty subjects agreed to complete the diagnosis and an additional 21 subjects agreed to family screening. We detected 9 cases with severe and 26 with intermediate A1ATD. Parallel experiments revealed that polymerization of M-type A1AT, when measured by nephelometry or isolelectrofocusing, yields inaccurate results, leading to the erroneous impression that it was Z type and not M-type A1AT. We illustrate the need for confirmation of Z A1AT values by “state of the art” method. Clinicians should consider a more in-depth investigation of A1ATD in patients when they exhibit serum polymers and low α1-globulin protein levels by SPE.

Correction: Comparison of Two Devices and Two Breathing Patterns for Exhaled Breath Condensate Sampling

The authors would like to amend this article based on the discovery of a number of errors that came to light after publication. We recognized a number of errors that made it necessary to repeat a part of the experiments. Please view the corrected text, table and figures here.