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Cancer research involves significant animal consumption and suffering. Tumor cells can be differentiated in vitro into three-dimensional organoids that resemble the primary tumor. In basic cancer research, however, tumor organoids are usually only used alongside animal experiments. We have established an easy-to-perform protocol that allows to culture KRAS-driven lung tumor cells as organoids for extended periods of time. Like the corresponding tumors in mice, the organoids produce surfactant protein C but no markers of airway epithelial cells (e.g. SCGB1A1, KRT5). The organoids can be passaged as single cell suspensions. Our organoid model contributes to replace animal experiments with cell culture systems and can be used for drug testing or functional studies in cancer research. [ABSTRACT FROM AUTHOR]

Abundant microbial colonization is a hallmark of COPD and smoke exposure likely increases the susceptibility to colonization and infection. The aim of the present study was to characterize the pulmonary changes of a combined exposure to cigarette smoke (CS) and microbial challenge in a preclinical murine COPD model. Animals were exposed to CS for 2 weeks, 3, and 6 months. Low and high doses of heat inactivated nontypeable Haemophilus influenzae (NTHi) were administered by inhalation during the whole exposure time. Pulmonary changes were analyzed by stereology, pulmonary function tests, measurements of inflammatory cells and mediators, and histopathology. Exposure of smoke in a relatively low concentration caused COPD-like changes of pulmonary function and only little inflammation. The coadministration of low dose NTHi (ld-NTHi) augmented a macrophage dominated inflammatory profile, while high dose NTHi (hd-NTHi) induced a neutrophilic inflammatory pattern. IL-17A secretion was solely dependent on the exposure to NTHi. Also goblet cell metaplasia and the formation of lymphoid aggregates depended on exposure to bacteria. In conclusion, the combination of exposure to smoke and bacterial compounds resulted in a mouse model that resembles several aspects of human disease. Exposure to microbial structural components appears necessary to model important pathologic features of the disease and the quantity of the exposure with microorganisms has a strong effect on the phenotype. [ABSTRACT FROM AUTHOR]

The activation of inflammasome signaling mediates pathology of acute Pseudomonas aeruginosa pneumonia. This suggests that the inflammasome might represent a target to limit the pathological consequences of acute P. aeruginosa lung infection. Pannexin-1 (Px1) channels mediate the activation of caspase-1 and release of IL-1β induced by P2X7 receptor activation. The approved drug probenecid is an inhibitor of Px1 and ATP release. In this study, we demonstrate that probenecid reduces infection and inflammation in acute P. aeruginosa pneumonia. Treatment of mice prior to infection with P. aeruginosa resulted in an enhanced clearance of P. aeruginosa and reduced levels of inflammatory mediators, such as IL-1β. In addition, probenecid inhibited the release of inflammatory mediators in murine alveolar macrophages and human U937 cell-derived macrophages upon bacterial infection but not in human bronchial epithelial cells. Thus, Px1 blockade via probenecid treatment may be a therapeutic option in P. aeruginosa pneumonia by improving bacterial clearance and reducing negative consequences of inflammation. [ABSTRACT FROM AUTHOR]

Background: Alpha1-antitrypsin (AAT) deficiency, although largely under-diagnosed, is the underlying cause of approximately 1% of COPD cases. Lack of awareness leads to long delays in diagnostic testing. Subsequently, lifestyle and treatment choices with potentially positive effects on prognosis may be postponed. Methods: Data on the testing and diagnostic practices for AAT deficiency were derived from the University of Pavia, Italy, and the University of Marburg, Germany. In addition, a survey of physicians was undertaken to explore their awareness and attitudes toward AAT deficiency. Results: In Pavia and Marburg, 125 and 729 patients, respectively, were identified with severe AAT deficiency between July 2006 and June 2011. The median time interval between the onset of symptoms and diagnosis was 6 years (interquartile range [IQR], 11; range, 0-40) and 7 years (IQR, 13; range, 0-73), respectively. Augmentation therapy was initiated almost immediately in Germany while treatment was delayed by 3 months in Italy (IQR, 5.25; range, 1-118). Survey data (Italy, n = 181; Germany, n = 180) revealed that pulmonologists had greater knowledge of AAT deficiency than internists and general practitioners, however, overall, only 18-25% of physicians tested all COPD patients. One-third of the respondents stated that they "sometimes" offered augmentation therapy to patients diagnosed with AAT deficiency. Conclusions: Major obstacles to AAT deficiency testing are physicians' attitudes and lack of understanding of the condition. A greater adherence to the guidelines that recommend diagnostic testing of all COPD patients, coupled with simpler testing protocols, may decrease delays and positively impact patient outcomes.protocols, may decrease delays and positively impact patient outcomes. [ABSTRACT FROM AUTHOR]

Summary: Background: Exacerbations of chronic obstructive pulmonary disease (COPD) significantly contribute to COPD-related morbidity. Diagnosis of COPD exacerbations may be improved by analyzing biomarkers such as alpha-1 antitrypsin (AAT). AAT is an acute-phase protein and inhibitor of neutrophil elastase. Deficiency of AAT may result in early-onset respiratory symptoms. Measurement of exhaled breath condensate (EBC) is a noninvasive method to investigate biomarkers present in the epithelial lining fluid, such as AAT. Objective: To investigate whether AAT can be detected and quantified in EBC and to compare AAT levels in the EBC of healthy controls, patients with COPD, and during exacerbations of COPD. Methods: EBC from 10 healthy controls, 17 subjects with COPD, and 18 subjects with exacerbations of COPD was collected with the RTube™ device. AAT from EBC and serum were quantified by ELISA. Results: AAT in EBC was detectable in every individual. Patients with exacerbations of COPD had significantly increased AAT values (mean, 514.33pg/mL, [SD 279.41 ]) compared with healthy controls (mean, 251.32pg/mL, [SD 44.71]) and stable COPD patients (mean, 242.01pg/mL [SD 65.74]) (P =0.0003; P =0.00003). EBC AAT showed only a correlation trend with serum AAT (r =0.3, P =0.054). Conclusions: AAT in EBC was detectable and quantifiable. AAT measured in EBC was significantly increased during exacerbations of COPD and can potentially be used as a biomarker in exacerbations. [Copyright &y& Elsevier]

Summary: α1-Antitrypsin (AAT) is the archetype member of the serine protease inhibitor (SERPIN) supergene family. The AAT deficiency is most often associated with the Z mutation, which results in abnormal Z AAT folding in the endoplasmic reticulum of hepatocytes during biogenesis. This causes intra-cellular retention of the AAT protein rather than efficient secretion with consequent deficiency of circulating AAT. The reduced serum levels of AAT contribute to the development of chronic obstructive pulmonary disease (COPD) and the accumulation of abnormally folded AAT protein increases risk for liver diseases. In this review we show that with the discovery of AAT deficiency in the early 60s as a genetically determined predisposition to the development of early-onset emphysema, intensive investigations of enzymatic mechanisms that produce lung destruction in COPD were pursued. To date, the role of AAT in other than lung and liver diseases has not been extensively examined. Current findings provide new evidence that, in addition to protease inhibition, AAT expresses anti-inflammatory, immunomodulatory and antimicrobial properties, and highlight the importance of this protein in health and diseases. In this review co-occurrence of several diseases with AAT deficiency is discussed. [ABSTRACT FROM AUTHOR]

Alpha-1-antitrypsin deficiency (AATD) is a rare genetic disorder associated with the development of liver and lung disease. AAT is a 52-kD glycoprotein, produced mainly by hepatocytes and secreted into the blood. Agglomeration of the AAT-protein in hepatocytes can result in liver disease. Exposure to smoke is the major risk factor for the development of lung disease characterised as early chronic obstructive lung disease (COPD). Diagnosis is based on the analysis of the AAT genotype and phenotype. The measurement of the AAT serum level is useful as screening test. Liver biopsy is not necessary to establish the diagnosis. Therapy for AAT-related liver disease is supportive, a specific therapy is not available. AATD is a rare condition (1:5000–10000) and, as a consequence, data and information on diagnosis and treatment are not easily accessible. This chapter provides a comprehensive overview on AATD, covering basic biology, diagnostic and therapeutic approaches. [ABSTRACT FROM AUTHOR]

Summary: In the last years an increasing number of subjects experienced respiratory failure and underwent tracheostomy. The aim of the present study was to analyze tracheal aspirates from the inflammatory point of view. Samples were collected from 38 consecutive tracheostomized patients: 13 COPD, 6 with neurologic disorders and 19 with other different causes of respiratory failure. We analyzed cells and soluble mediators related to inflammation and/or infection. We also compared results obtained in the tracheal aspirate of COPD patients with the same determination in the sputum of another group of non-tracheostomized COPD patients (n =41). Regardless of the underlying diagnosis, most of the samples were Pseudomonas aeruginosa positive and cells and soluble mediator did not differ. Treatment with steroids was associated with lower amount of total cells, neutrophils and lymphocytes, whereas treatment with antibiotics was not. Tracheal aspirate neutrophils correlated with PaCO2 values; neutrophils and eosinophils correlated with their percentages in blood. As compared with sputa obtained from another group of culture-positive non-tracheostomized COPDs, tracheal aspirates showed similar cell count, proportions of inflammatory cells, and infection/inflammatory mediators. In conclusion tracheal aspirates presented high amounts of viable cells and soluble mediators independently to the cause of respiratory failure leading to tracheotomy and they represent suitable specimens to study infection/inflammation interactions. [Copyright &y& Elsevier]

Summary: The human gland of Moll located at the margin of the eyelids is a specialized apocrine gland, the function of which is not exactly known. The presence of antimicrobial proteins was identified in this gland recently, suggesting a function in the external ocular defense barrier against pathogens. In this study, we have demonstrated β-defensin-1, β-defensin-2 and cathelicidin (LL-37) in the secretory endpieces of the glands of Moll using immunohistochemical methods. β-Defensin-1, β-defensin-2 and cathelicidin (LL-37) showed a weak to moderately intensive staining pattern. The strongest immunolocalization of β-defensin-1 was observed in the apical protrusions of the gland, which could also be observed but to a lesser extent in the case of β-defensin-2 and cathelicidin. In active glandular cells, a granular staining pattern could be observed. β-Defensin-1 and β-defensin-2 varied in staining intensities, and even within one section strongly and weakly stained cells can coexist side by side. Also cells that, according to morphological criteria, appeared to be inactive still had an apical β-defensin-1 immunolabeling. We assume that β-defensin-1, β-defensin-2 and cathelicidin (LL-37) work together with other antimicrobial peptides and proteins to create a defensive barrier against microbial invasion at the ocular surface. [Copyright &y& Elsevier]

Summary: Background: Alpha-1-antitrypsin deficiency (AATD) is significantly underdiagnosed. The early detection of AATD would enable affected persons to make lifestyle changes such as quitting smoking. It was the aim of the study to determine whether the combination of an awareness program with the offer of a cost-free diagnostic test results in the identification of a significant number of individuals with severe AATD. Methodology: We combined a series of measures to promote awareness with the offer of a diagnostic test at no charge. Test blood was applied to a filter paper and sent to our laboratory. The level of AAT was measured by nephelometry, the presence of the S- or Z-allele was determined by PCR, and phenotyping was performed by isoelectric focusing. Results: During 37 months 17688 testing kits were distributed and 2722 were sent back to our laboratory. We identified 335 patients with severe AATD including 16 individuals with rare genotypes. Prescreening by determining the AAT serum levels by the submitting physician increased the detection rate as compared to similar programs that screened unselected individuals. Summary: These data show that the combination of an awareness program with the offer of free diagnostic testing results in the identification of a large number individuals with severe AATD. [Copyright &y& Elsevier]

A vast majority of the elderly population shows a mild to moderate vitamin D deficiency. Besides the well-known function of vitamin D, vitamin D receptor is also expressed in brain and is discussed to regulate several genes. However very little is known whether genes are regulated, associated with Alzheimer's disease (AD). Here we investigate 117 genes, known to be affected in AD, in mouse brain samples with a mild vitamin D hypovitaminosis comparable to the vitamin D status of the elderly population (20%-30% deficiency). The 117 genes include two positive controls, Nep and Park7, already known to be affected by both AD and vitamin D hypovitaminosis. The 25 most promising candidates were verified in a second independent mouse cohort, resulting in eleven genes further evaluated against three additional housekeeping genes. Three of the remaining eight significantly altered genes are involved in APP homeostasis (Snca, Nep, Psmb5), and each one gene in oxidative stress (Park7), inflammation (Casp4), lipid metabolism (Abca1), signal transduction (Gnb5) and neurogenesis (Plat). Our results tighten the link of vitamin D and AD and underline that vitamin D influences several genes also in brain, highlighting that a strong link not only to AD but also to other neurodegenerative diseases might exist. [ABSTRACT FROM AUTHOR]

Insufficient cerebral blood flow regulation to meet increasing metabolic demand during physical exertion could be associated with cognitive impairment. We compared cerebral oxygen availability during exercise in cognitively impaired (CI) to cognitively normal (CN) COPD patients. Fifty-two patients (FEV 1 : 51 ± 16%) were classified as CN or CI according to the Montreal Cognitive Assessment. Patients performed cycle-ergometry at 75% peak capacity with continuous measurement of Near-Infrared Spectroscopy frontal-cortex Tissue oxygen Saturation Index (TSI), cerebral haemoglobin indices (oxy/deoxy/total- Hb), transcutaneous carbon-dioxide partial pressure (TcPCO 2 ), and arterial oxygen saturation (SpO 2 ). Twenty-one patients (40%) presented evidences of CI. During exercise, CN and CI patients exhibited mild to moderate SpO 2 decline (nadir[Δ]≥ −3 ± 2% and −5 ± 3%, respectively) but preserved baseline frontal-cortex TSI levels, whilst presenting small TcPCO 2 perturbations and increased cerebral total-Hb (post [Δ]≥ 2.0 ± 3 μM sec −1 ). CI patients preserve the capacity to adequately maintain cerebral oxygen availability during submaximal exercise. Therefore, rehabilitative exercise training in CI patients with COPD exhibiting mild to moderate exercise-induced SpO 2 decline does not appear to lead to reduced cerebral oxygen availability. [ABSTRACT FROM AUTHOR]

The influence of vitamin D3 and its metabolites calcifediol (25(OH)D) and calcitriol on immune regulation and inflammation is well described, and raises the question of potential benefit against bacterial infections. In the current study, 25(OH)D was encapsulated in liposomes to enable aerosolisation, and tested for the ability to prevent pulmonary infection by Pseudomonas aeruginosa . Prepared 25(OH)D-loaded liposomes were nanosized and monodisperse, with a negative surface charge and a 25(OH)D entrapment efficiency of approximately 23%. Jet nebulisation of liposomes was seen to yield an aerosol suitable for tracheo-bronchial deposition. Interestingly, 25(OH)D in either liposomes or ethanolic solution had no effect on the release of the proinflammatory cytokine KC from Pseudomonas -infected murine epithelial cells (LA-4); treatment of infected, human bronchial 16-HBE cells with 25(OH)D liposomes however resulted in a significant reduction in bacterial survival. Together with the importance of selecting an application-appropriate in vitro model, the current study illustrates the feasibility and practicality of employing liposomes as a means to achieve 25(OH)D lung deposition. 25(OH)D-loaded liposomes further demonstrated promising effects regarding prevention of Pseudomonas infection in human bronchial epithelial cells. [ABSTRACT FROM AUTHOR]

Extracorporeal membrane oxygenation (ECMO) has been used successfully for the treatment of patients with respiratory failure due to severe infections. Although rare, parasites can also cause severe pulmonary disease. Tapeworms of the genus Echinococcus give rise to the development of cystic structures in the liver, lungs, and other organs. Acute cyst rupture leads to potentially life-threatening infection, and affected patients may deteriorate rapidly. The case of a young woman from Bulgaria who was admitted to hospital with severe dyspnoea, progressive chest pain, and haemoptysis is described. Computed tomography of the chest was pathognomonic for cystic echinococcosis with acute cyst rupture. Following deterioration on mechanical ventilation, she was cannulated for veno-venous ECMO. The patient’s condition improved considerably, and she was weaned successfully from ECMO and mechanical ventilation. Following lobectomy of the affected left lower lobe, the patient was discharged home in good condition. This appears to be the first report of the successful use of ECMO as salvage treatment for a severe manifestation of a helminthic disease. Due to recent migration to Western Europe, the number of patients presenting with respiratory failure due to pulmonary echinococcosis with cyst rupture is likely to increase. [ABSTRACT FROM AUTHOR]

Carbon metabolism and virulence determinant production are often linked in pathogenic bacteria, and several regulatory elements have been reported to mediate this linkage in Staphylococcus aureus. Previously, we described a novel protein, catabolite control protein E (CcpE) that functions as a regulator of the tricarboxylic acid cycle. Here we demonstrate that CcpE also regulates virulence determinant biosynthesis and pathogenesis. Specifically, deletion of ccpE in S. aureus strain Newman revealed that CcpE affects transcription of virulence factors such as capA, the first gene in the capsule biosynthetic operon; hla, encoding α-toxin; and psmα, encoding the phenol-soluble modulin clusterα. Electrophoretic mobility shift assays demonstrated that CcpE binds to the hla promoter. Mice challenged with S. aureus strain Newman or its isogenic ΔccpE derivative revealed increased disease severity in the ΔccpE mutant using two animal models; an acute lung infection model and a skin infection model. Complementation of the mutant with the ccpE wild-type allele restored all phenotypes, demonstrating that CcpE is negative regulator of virulence in S. aureus. [ABSTRACT FROM AUTHOR]

Abstract: Coronary artery disease (CAD), chronic heart failure (CHF) or chronic obstructive pulmonary disease (COPD) occur commonly in the presence of each other and are associated with similar systemic inflammatory reactions. Inflammation plays a central role in the pathogenesis of these diseases. C-reactive protein (CRP) could represent the sentinel biomarker to all chronic diseases. Also, interleukin (IL)-6 may play a causal role in systemic inflammatory diseases. These complex interactions between heart and lung can be denoted as ‘cardiopulmonary continuum’. Common risk factors induce systemic inflammatory processes which lead to progression of atherosclerotic diseases as well as COPD. [ABSTRACT FROM AUTHOR]

Summary: Cancer development can be viewed as dysregulated repair. Antimicrobial peptides (AMPs) are effector molecules of the innate immune system with direct antimicrobial activity. Beside this host defence function several AMPs play a role in the regulation of inflammation and tissue repair. The aim of the present study was to investigate whether the human cathelicidin AMP LL-37/hCAP-18 is involved in the biology of lung cancer. Human cancer cell lines were found to express the human cathelicidin LL-37/hCAP-18 mRNA and peptide at different levels. Immunohistochemistry of human lung cancers showed that the peptide is expressed mostly in adenocarcinoma and squamous cell carcinoma. Application of exogenous LL-37 at low concentrations of 5ng/ml to cancer cell lines increased proliferation and growth of anchorage-independent colonies. At the molecular level, LL-37 induced phosphorylation of the epidermal growth factor receptor (EGFR) and activation of downstream MAP kinase signalling pathways. Lung cancer cell lines that stably overexpress the peptide by means of a doxycycline-regulated promoter system also showed a faster growth. When these cell lines were injected subcutaneously into nude mice, cathelicidin overexpression resulted in increased tumourigenicity and the formation of significantly larger tumours. In conclusion, cathelicidin is expressed in human lung cancers. The peptide activates tumour cells resulting in increased cell growth in vitro and in an animal model. The host defence peptide cathelicidin LL-37/hCAP-18 acts as growth factor for human lung cancer. [Copyright &y& Elsevier]

Abstract: Antimicrobial peptides (AMPs) and mucins are components of airway secretions and both contribute to the innate host defense system. At neutral pH, AMPs are positively charged, mucins negatively. It was the aim of the study to test whether these opposite charges result in interactions between AMPs and mucins. We measured binding of mucins isolated from porcine gastric mucosa to the cathelicidin LL-37 coated to multiwell plates and found that LL-37 electrostatically interacts with mucins. Circular dichroism spectra of the peptide revealed the induction of α-helical conformation by mucins. Addition of mucins to solutions of LL-37 significantly decreased the antimicrobial activity of the peptide against Pseudomonas aeruginosa and Streptococcus pneumoniae. We then tested whether LL-37 is bound to mucins in airway secretions from human subjects and found that a significant proportion of the peptide and its propeptide are bound to high molecular weight components. Together these data show that cationic AMPs interact with anionic mucins in airway secretions. Functions of AMPs are modulated by this interaction. [Copyright &y& Elsevier]

NF-κB mediated inflammation is a key process to many diseases. RNA interference (RNAi) is the specific suppression of genes by short double-stranded RNA. It was the aim of the present study to modify NF-κBdependent inflammation by small interfering RNA (siRNA) expressed by recombinant adeno-associated virus (rAAV). To study the kinetics of rAAV mediated expression of siRNA, the expression of the luciferase gene was targeted and resulted in a significant decrease of luciferase activity as compared to a control vector in the human 293 cell line. The effect was dose dependent and was detectable 24 h after infection. rAAV coding for siRNA against the p65 subunit of NF-κBsignificantly reduced the p65 protein. In a cellular model of TNF-α induced inflammation, expression of siRNA against p65 significantly suppressed the secretion of IL-8 from BEAS-2B cells. In conclusion, rAAV vectors coding for siRNA are an useful tool for efficient gene silencing in mammalian cells and can be used to modify NF-κB mediated inflammation. [Copyright &y& Elsevier]