Ruth Sepper

In vivo collagenase‐2 (MMP‐8) expression by human bronchial epithelial cells and monocytes/macrophages in bronchiectasis

The purpose of this study was to determine whether other cellular sources than neutrophils can express matrix metalloproteinase (MMP)‐8 protein and mRNA in bronchiectatic (BE) lung. The molecular forms of MMP‐8 in the BE bronchoalveolar lavage fluid (BALF) and healthy control BALF were analysed by western immunoblotting. MMP‐8 expression was demonstrated by immunohistochemistry and in situ hybridization in BE lung tissue and by immunohistochemistry in control lung tissue. In the BE BALF, different MMP‐8 species were detected: 70–80 kD MMP‐8 apparently of polymorphonuclear leukocyte (PMN) origin and also 40–60 kD MMP‐8 from non‐PMN cellular sources, such as bronchial epithelial cells, glandular cells or monocytes/macrophages. Both of these MMP‐8 species were elevated and converted to a significant extent to activated forms in BE BALF compared with healthy control BALF. The levels of high molecular weight (>80 kD) MMP‐8 complexes, evidently representing MMP‐8 trapped by endogenous MMP inhibitors and/or MMP‐8 dimers, were significantly elevated in BE BALF compared with healthy control BALF. In BE lung tissue, the MMP‐8 protein and mRNA expression was found in bronchial ciliated epithelial cells, glandular cells, neutrophils, and monocytes/macrophages infiltrating the bronchial epithelial area. Minimal MMP‐8 expression was observed in neutrophils, monocytes/macrophages, and epithelial cells in control lung tissues. In this study, new potential cellular sources have been demonstrated for MMP‐8 in the inflamed lung. MMP‐8 from multiple cellular sources, including inflamed lung epithelium, was activated to a significant extent in the BE BALF, indicating a major role for MMP‐8 in the destruction of lung and bronchial tissues. Copyright

Airway obstruction correlates with collagenase-2 (MMP-8) expression and activation in bronchial asthma.

Matrix metalloproteinases (MMPs) contribute to extracellular matrix and basement membrane degradation in asthma. The present study analyzed molecular forms and degree of activation and expression of MMP-8 in bronchoalveolar lavage fluid (BALF), BALF cells, and bronchial tissue specimens from 14 steroid-naive asthma patients, 13 uncontrolled severe asthma patients, 13 controlled asthma patients, and 14 healthy subjects by Western immunoblotting, immunohistochemistry, and in situ hybridization. Immunohistochemistry and in situ hybridization revealed a prominent MMP-8 immunoreactivity in submucosal inflammatory, glandular, and shed, but not in intact bronchial epithelial cells of asthma patients. In BALF cytospins, both MMP-8 protein and mRNA expression were observed in epithelial cells, macrophages, and polymorphonuclear leukocytes (PMNs). MMP-8 was present in BALFs asthma patients in complex, pro- and active PMN-type, and pro- and active non-PMN–type forms. BALF MMP-8 was significantly converted to active form only in BALFs from steroid-naive and uncontrolled severe asthma patients, but not in BALFs from well-controlled asthma patients or healthy controls. A significant inverse correlation between BALF MMP-8 levels and FEV1 (r = −0.283, p = 0.04), and BALF activated MMP-8 forms and FEV1 (r = −0.427, p = 0.001) was detected. Overall, these data suggest that MMP-8 and its activation has an important role in the airway destruction, healing, remodeling, and treatment response in asthma.

Direct demonstration of tissue uptake of an inhaled drug: proof-of-principle study using matrix assisted laser desorption ionization mass spectrometry imaging

Drug therapy is often directed to specific organ and tissue compartments where the mode of action of the compound affects specifically targeted biological processes. However, the direct measurement of drug uptake in terms of a time kinetic and concentrations attained at the local sites has not been readily available as a clinical index for most drugs. A proof-of-principle study was conducted to test the utility of applying matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) to demonstrate the qualitative distribution pattern of a locally administered drug within tissue sites of targeted action. Here we have measured the occurrence of an inhaled bronchodilator, the muscarinic receptor antagonist ipratropium, within human bronchial biopsies obtained by fiber optic bronchoscopy shortly after dosing exposure. Cryo-preserved biopsy samples from five subjects being evaluated for airway obstruction or potential tumor development were prepared as thin frozen sections. Samples coated with a MALDI matrix were analyzed by a MALDI LTQ Orbitrap XL mass spectrometer at large (100 μm) and small (30 μm) raster sizes. Our results demonstrate that ipratropium is rapidly absorbed into the airway wall. Ipratropium parent ion (m/z 332.332) and daughter ions (m/z 166.2 and 290.2) were coincidently partitioned within submucosal spaces containing targeted airway smooth muscle in four out of five subjects. The signal intensity of ipratropium fragment ions provided estimates that local drug concentrations between 3 and 80 nM were achieved within the airway wall. To our knowledge, this is the first reported study in applying MALDI-MSI to demonstrate the localization of a drug administered at therapeutic levels. The study highlights the potential benefit of MALDI-MSI to provide important measurements of drug efficacy in clinical settings.

Soluble membrane‐type 1 matrix metalloproteinase (MT1‐MMP) and gelatinase A (MMP‐2) in induced sputum and bronchoalveolar lavage fluid of human bronchial asthma and bronchiectasis

The aim of this study was to investigate the involvement of the MT1‐MMP/MMP‐2 cascade in induced sputum (IS) and bronchoalveolar lavage fluid (BALF) from bronchial asthma (BA) and bronchiectasis (BE) patients and healthy controls. The molecular forms and cellular origins of MT1‐MMP and MMP‐2 were determined by Western immunoblotting, immunohistochemistry and in situ hybridization. Elevated levels of soluble activated and autocatalyzed MT1‐MMP species as well as activated forms of MMP‐2 in IS and BALF samples from BA and BE patients were evidenced. The activation degrees of soluble MT1‐MMP and MMP‐2 were significantly correlated in BA and BE IS and BALF. Only low levels of both these MMPs were observed in healthy control IS and BALF. The co‐expression of MMP‐2 with MT1‐MMP was evidenced by double immunostaining in bronchial epithelial cells, submucosal glandular cells, smooth muscle cells and monocyte/macrophages. The MT1‐MMP/MMP‐2 cascade is present and active in human inflammatory lung disease fluid and tissue samples. This cascade seemingly reflects the active destructive phases of these chronic lung diseases.

Understanding drug uptake and binding within targeted disease micro-environments in patients: a new tool for translational medicine.

BackgroundFor many common global diseases, such as cancer, diabetes, neurodegenerative and cardiovascular diseases there is an unmet need for diagnosing early indications of disease that could enable medical intervention and early treatment. The treatment of these diseases will require detailed knowledge of targeted pathways involved in disease pathogenesis but also the mode of drug actions at the biological location on these targets. Translational medicine is a new area of research where expert from different disciplines involved in basic science and clinical disciplines meet and join forces. Mode-of-drug-action mechanisms elucidation is key in the characterization of drugs that can relate to both efficacy and safety.MethodsMatrix assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used providing evidence into the fate (destinations and distributions) of administered drugs within tumor regions of lung compartments.ResultsWe hereby present a pulmonary study in which we have isolated lung tissue after inhaled drug administration and then localized the drug within airway wall compartments. The histology also provides evidence of drug binding to smooth muscle cell microenvironments. We also identified lung tissue regions with tumor cell invasion in these COPD patients.ConclusionsThe ultimate goal is to identify bridging comprehension that forms a knowledge base that can be used by society to develop a better treatment and medicine for patients. Our results demonstrated that robust imaging data could be generated confirming drug localization in pulmonary regions of COPD patients with tumor pathology.Trial registrationTallinn Medical Research Ethical Committee decision #1724, 18.06.2009

Association of trypsin-2 with activation of gelatinase B and collagenase-2 in human bronchoalveolar lavage fluid in vivo

BACKGROUND: Tissue injury mediated by matrix metalloproteinases (MMPs) is a hallmark of inflammatory lung diseases. Latent secreted proMMPs must be activated to be catalytically competent. AIM: Our aim was to analyse an involvement of the trypsin-2, trypsin-2-α1-proteinase inhibitor (PI) complex and tumour-associated trypsin inhibitor (TATI) in the in vivo activation of proMMP-8, -9 and -2. METHODS: Concentrations of trypsin-2, trypsin-Z-a,-PI complex and TAT1 in bronchoalveolar lavage fluid (BALF) were analysed by immunofluorometry. Molecular forms and expression of trypsin-2 and trypsin-2-α,-PI complex were identified by Western immunoblot and immunocyto-chemistry. Gelatinolytic and collagenolytic activities were measured by substrate-based activity assays. RESULTS: BALFs from 16 of 43 patients and BALFs from five of 15 healthy controls contained trypsin-2-a,-PI complex. TAT1 was found in all healthy control BALFs (median 0.12 μg/L, range 0.02-0.66 μg/L) whereas 8 of 43 BALFs from patients (median 0, range 0-0.64 μg/L, P = 0.0001) contained TATI. Patient BALFs showed significantly increased activation of MMP-9 and MMP-8 compared with healthy controls. The concentrations of trypsin-2-α,-PI complex correlated with the in vivo activation of MMP-9 and -8 (r = 0.68, P= 0.002 and r = 0.61, P = 0.008) but not with the activation of MMP-2 in BALFs.

Lung Dysfunction of Chronic Smokers with No Signs of COPD

Cigarette smoking causes airflow limitation with lung hyperinflation being the primary causes of COPD. Fifty chronic smokers (CSs) with no signs of GOLD-adjusted COPD with smoking habit at least ≥10 pack-years (p/yrs) were divided into CS-mild (n = 24) with smoking history from ≥10 to ≤20 p/yrs and CS-heavy groups (n = 26) with smoking history ≥21 p/yrs. Spirometry, plethysmography and diffusing capacity were measured and lung computed tomography (CT) was performed. Residual volume (RV) (L) and RV/TLC (total lung capacity) ratio were significantly increased in CS-heavy when compared to CS-mild (p = 0.001, p = 0.03). A significant reduction of forced expiratory volume in 1 second/forced vital capacity (FEV1/FVC) ratio and airway specific conductance was shown in CS-heavy (p = 0.02, p = 0.03). Lung emphysema signs at CTs were revealed in 17 CSs and ten of them had declined diffusing capacity below 70% of predicted. The percentage of emphysematous lesions inversely and significantly correlated with measured diffusing capacity (p = 0.0009, r = −0.72). Study groups’ smoking intensity inversely correlated the declined airway specific conductance (p = 0.004, r = −0.39) and increase of the RV (L) (p = 0.0004, r = 0.46). Multiple regression analysis determined that smoking intensity regardless of the subjects’ age was significant factor for decline of airway specific conductance and increase of RV (L). Here we conclude that lung function deviation and lung structural changes are present in CSs before the clinical signs of airway obstruction reveal. Body plethysmography and diffusing capacity measurement with routine spirometry can provide valuable information for detection of changes reflecting to the early onset of COPD in CSs.

Mast cells in bronchiectasis

Bronchiectasis (BE) is a chronic severe inflammatory lung disease characterized by frequent bacterial infections and polymorphonuclear neutrophil-dominated inflammatory reaction. We have attempted to elucidate the role of mast cells (MCs) in BE lung inflammation by measuring in the bronchoalveolar lavage fluid (BALF) the MC-derived tryptase levels by radioimmunoassay and immunoblotting and also by measuring the tryptase-like activities in 36 BE patients and in 14 healthy controls. The amount of MC in the lung tissue was assessed by immunohistochemical staining of resected lung tissue samples. Based on the clinical and radiological parameters the patients were divided into subgroups according to the severity of the disease. The MC tryptase concentrations (microg/L; median (range)) in BALF of BE patients were higher compared to healthy controls (4.7(1.4-20.1) and 2.0 (0.1-3.5), respectively, P < 0.01). Tryptase concentrations in the groups of mild, moderate and severe BE were 3.8 (0.9-10.8), 4.3 (3.0-12.6) and 9.6 (1.2-20.1), respectively. All the values differed significantly from those observed in the healthy controls. The tryptase-like activities (nmol/sec/L) in BE patients were also markedly increased (174 (31-2874)) compared to healthy controls (28 (9-45) P < 0.0001). The tryptase-like activities in the patient subgroups were 45 (36-598) in mild, 91 (31-1437) in moderate and 1336 (37-2874) in severe BE. Again, all values differed significantly from those observed in the healthy controls. Moreover, immunoblot experiments disclosed the most intensive immunoreactivity of the 27.5 kD tryptase monomer in BALF of patients with severe BE followed by weaker immunoreactivity in groups of moderate and mild BE and in healthy controls. No significant difference could be observed in the amount of tryptase-positive cells between BE patients and controls. However, the presence of degranulated MCs was more evident in BE lung tissue. Significant correlation could also be observed between the degree of activation of latent procollagenase and tryptase concentration (r = 0.8, P = 0.0004) in BALF of individual BE patients. The observed strong correlation between tryptase levels and disease severity suggests that MCs may be involved in the inflammatory reaction in the BE lung. Importantly, the high levels of tryptase, observed also in patients with mild BE, suggests that activation of and proteinase release from MCs may be one of the reasons for the perpetuation of tissue injury even during the clinically quiescent periods in BE.

Nationwide Health Data Management System: a novel approach for integrating biomarker measurements with comprehensive health records in large populations studies.

The nation-wide electronic health record database acts as an interoperable repository of health data obtained throughout citizen contacts with health care providers. This system improves accessibility for citizens and researchers to health data with the ability to assign context to disease development. In that system, individual patients who are members of the large population-based health database can be assessed as individuals or as a population in prospective studies of prospective diseases.

Role of politics in public sector organizational change

In this paper we will address issues of organizational changes in public sector where the relevance of management has not diminished during the last two decades of the neo‐liberal market philosophy. Public sector organizations are susceptible to greater and more open accountability with politicians, pressure groups, taxpayers and voters all having an interest in the performance of it. In late 1990s Estonian government initiated reforms of health care system in the country. Estonian Hospital Master Plan (EHMP) 2015 was launched in 2000 which, within the others, was initiating the merge of seven Tallinn hospitals into North Estonian Regional Hospital (NERH). To evaluate efficacy of organizational changes during implementation of EHMP‐model into health care system in Estonia we utilized personal interviews of top and middle managers and annual reports of merged hospitals to benchmark these measures to earlier merged Univerity Hopsital and other EU hospitals. We conclude that even NERH was established and the reform‐initiated changes were mostly introduced by the deviation from first‐line governmental plans and introduction of new political directions in 2003 lead to new organizational changes and managerial efforts to gain the goals with, unfortunately, prolonged change process.