Helene Skjøt-Arkil

Measurement of MMP-9 and -12 degraded elastin (ELM) provides unique information on lung tissue degradation

BackgroundElastin is an essential component of selected connective tissues that provides a unique physiological elasticity. Elastin may be considered a signature protein of lungs where matrix metalloprotease (MMP) -9-and -12, may be considered the signature proteases of the macrophages, which in part are responsible for tissue damage during disease progression. Thus, we hypothesized that a MMP-9/-12 generated fragment of elastin may be a relevant biochemical maker for lung diseases.MethodsElastin fragments were identified by mass-spectrometry and one sequence, generated by MMP-9 and -12 (ELN-441), was selected for monoclonal antibody generation and used in the development of an ELISA. Soluble and insoluble elastin from lung was cleaved in vitro and the time-dependent release of fragments was assessed in the ELN-441 assay. The release of ELN-441 in human serum from patients with chronic obstructive pulmonary disease (COPD) (n = 10) and idiopathic pulmonary fibrosis (IPF) (n = 29) were compared to healthy matched controls (n = 11).ResultsThe sequence ELN-441 was exclusively generated by MMP-9 and -12 and was time-dependently released from soluble lung elastin. ELN-441 levels were 287% higher in patients diagnosed with COPD (p < 0.001) and 124% higher in IPF patients (p < 0.0001) compared with controls. ELN-441 had better diagnostic value in COPD patients (AUC 97%, p = 0.001) than in IPF patients (AUC 90%, p = 0.0001). The odds ratios for differentiating controls from COPD or IPF were 24 [2.06–280] for COPD and 50 [2.64–934] for IPF.ConclusionsMMP-9 and -12 time-dependently released the ELN-441 epitope from elastin. This fragment was elevated in serum from patients with the lung diseases IPF and COPD, however these data needs to be validated in larger clinical settings.

Macrophage-Mediated Proteolytic Remodeling of the Extracellular Matrix in Atherosclerosis Results in Neoepitopes: A Potential New Class of Biochemical Markers

Worldwide, cardiovascular disease (CVD) is the leading cause of death. Most CVD-related deaths are caused by years of preceding atherogenesis and the extensive development of atherosclerotic plaques, some of which may rupture to cause myocardial infarction. Macrophages are known to have a role in almost all stages of atherosclerosis, by both initiating atherosclerotic plaques and degrading them through the secretion of proteolytic enzymes leading to rupture. This review summarizes the literature on the role of macrophages and their proteolytic activity on proteins in the extracellular matrix (ECM) of the atherosclerotic plaque with a view to suggest a novel approach for identification of vulnerable plaques and turnover by the use of a new type of biomarker. The PubMed database was searched using the terms macrophages, foam cells, atherosclerosis, CVD, ECM remodeling, biomarker, neoepitope, matrix metalloproteinase (MMP), and protease. Atherosclerotic plaques are primarily composed of the protein type I and III collagen, and smaller quantities of elastin and proteoglycans. Macrophages secrete an array of proteases, including MMPs, cathepsins, and aggrecanases, with the ability to degrade most of the constituents of the ECM of the atherosclerotic plaque. At present it is not clear which proteases play pivotal roles at distinct stages of pathogenesis, rather that the combined proteolytic potential with some proteases at early stages and other at later stages may result in plaque rupture. This macrophage-mediated proteolysis and remodeling of the ECM play important roles in many stages of atherosclerosis. The degradation fragments of these ECM events are specific neoepitopes, which are released into the circulation. The identification of these pathologically relevant neoepitopes leads to novel biomarkers able to identify the formation and degradation of plaques providing different biological information than traditionally used biomarkers.

Human macrophage foam cells degrade atherosclerotic plaques through cathepsin K mediated processes

BackgroundProteolytic degradation of Type I Collagen by proteases may play an important role in remodeling of atherosclerotic plaques, contributing to increased risk of plaque rupture.The aim of the current study was to investigate whether human macrophage foam cells degrade the extracellular matrix (ECM) of atherosclerotic plaques by cathepsin K mediated processes.MethodsWe 1) cultured human macrophages on ECM and measured cathepsin K generated fragments of type I collagen (C-terminal fragments of Type I collagen (CTX-I) 2) investigated the presence of CTX-I in human coronary arteries and 3) finally investigated the clinical potential by measuring circulating CTX-I in women with and without radiographic evidence of aortic calcified atherosclerosis.ResultsImmune-histochemistry of early and advanced lesions of coronary arteries demonstrated co-localization of Cathepsin-K and CTX-I in areas of intimal hyperplasia and in shoulder regions of advanced plaques. Treatment of human monocytes with M-CSF or M-CSF+LDL generated macrophages and foam cells producing CTX-I when cultured on type I collagen enriched matrix. Circulating levels of CTX-I were not significantly different in women with aortic calcifications compared to those without.ConclusionsHuman macrophage foam cells degrade the atherosclerotic plaques though cathepsin K mediated processes, resulting in increase in levels of CTX-I. Serum CTX-I was not elevated in women with aortic calcification, likely due to the contribution of CTX-I from osteoclastic bone resorption which involves Cathepsin-K. The human macrophage model system may be used to identify important pathway leading to excessive proteolytic plaque remodeling and plaque rupture.

Acute Myocardial Infarction and Pulmonary Diseases Result in Two Different Degradation Profiles of Elastin as Quantified by Two Novel ELISAs.

Background Elastin is a signature protein of the arteries and lungs, thus it was hypothesized that elastin is subject to enzymatic degradation during cardiovascular and pulmonary diseases. The aim was to investigate if different fragments of the same protein entail different information associated to two different diseases and if these fragments have the potential of being diagnostic biomarkers. Methods Monoclonal antibodies were raised against an identified fragment (the ELM-2 neoepitope) generated at the amino acid position ‘552 in elastin by matrix metalloproteinase (MMP) −9/−12. A newly identified ELM neoepitope was generated by the same proteases but at amino acid position ‘441. The distribution of ELM-2 and ELM, in human arterial plaques and fibrotic lung tissues were investigated by immunohistochemistry. A competitive ELISA for ELM-2 was developed. The clinical relevance of the ELM and ELM-2 ELISAs was evaluated in patients with acute myocardial infarction (AMI), no AMI, high coronary calcium, or low coronary calcium. The serological release of ELM-2 in patients with chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF) was compared to controls. Results ELM and ELM-2 neoepitopes were both localized in diseased carotid arteries and fibrotic lungs. In the cardiovascular cohort, ELM-2 levels were 66% higher in serum from AMI patients compared to patients with no AMI (p<0.01). Levels of ELM were not significantly increased in these patients and no correlation was observed between ELM-2 and ELM. ELM-2 was not elevated in the COPD and IPF patients and was not correlated to ELM. ELM was shown to be correlated with smoking habits (p<0.01). Conclusions The ELM-2 neoepitope was related to AMI whereas the ELM neoepitope was related to pulmonary diseases. These results indicate that elastin neoepitopes generated by the same proteases but at different amino acid sites provide different tissue-related information depending on the disease in question.

Tumor necrosis factor-α and receptor activator of nuclear factor-κB ligand augment human macrophage foam-cell destruction of extracellular matrix through protease-mediated processes.

By secreting proteases such as cathepsins and matrix metalloproteinases (MMPs), macrophage foam cells may be a major cause of ruptured atherosclerotic plaques. The aims of the present study were to investigate in vitro role of human macrophage foam cells in degrading type I collagen, a major component of extracellular matrix (ECM) in plaques, and to establish whether the pro-inflammatory molecules, tumor necrosis factor (TNF)-alpha, and receptor activator of nuclear factor-κB ligand (RANK-L) increase this degradation. CD14+ monocytes isolated from peripheral blood were differentiated into macrophage foam cells and cultured on a type I collagen matrix in the presence of TNF-alpha and RANK-L. Matrix degradation was measured by the cathepsin K-generated C-terminal cross-linked telopeptide of type I collagen (CTX-I) and the MMP-generated carboxyterminal telopeptide of type I collagen (ICTP) in supernatants showing that macrophage foam cells secrete MMPs and cathepsin K, resulting in release of ICTP and CTX-I. Stimulation with TNF-alpha increased CTX-I and ICTP dose dependently, with ICTP levels increasing by 59% and CTX-I levels increasing by 43%. RANK-L enhanced the release of CTX-I and ICTP by 56% and 72%, respectively. This is, to our knowledge, the first data describing a simple in vitro system in which macrophage foam cells degradation of matrix proteins can be monitored. This degradation can be enhanced by cytokines since TNF-alpha and RANK-L significantly increased the matrix degradation. This in vitro system in part is a model system for the macrophage-mediated proteolytic degradation of the ECM, which is found in many diseases with an inflammatory component.

Multifaceted Pharmacist-led Interventions in the Hospital Setting: A Systematic Review

Clinical pharmacy services often comprise complex interventions. In this MiniReview, we conducted a systematic review aiming to evaluate the impact of multifaceted pharmacist-led interventions in a hospital setting. We searched MEDLINE, Embase, Cochrane Library and CINAHL for peer-reviewed articles published from 2006 to 1 March 2018. Controlled trials concerning hospitalized patients in any setting receiving patient-related multifaceted pharmacist-led interventions were considered. All types of outcome were accepted. Inclusion and data extraction were performed. Study characteristics were collected, and risk of bias assessment was conducted utilizing the Cochrane Risk of Bias tools. All stages were conducted by at least two independent reviewers. The review was registered in PROSPERO (CRD42017075808). A total of 11,896 publications were identified, and 28 publications were included. Of these, 17 were conducted in Europe. Six of the included publications were multi-centre studies, and 16 were randomized trials. Usual care was the comparator. Significant results on quality of medication use were reported as positive in eleven studies (n = 18; 61%) and negative in one (n = 18, 6%). Hospital visits were reduced significantly in seven studies (n = 16; 44%). Four studies (n = 12; 33%) reported a positive significant effect on either length of stay or time to revisit, and one study reported a negative effect (n = 12; 6%). All studies investigating mortality (n = 6), patient-reported outcome (n = 7) and cost-effectiveness (n = 1) showed no significant results. This MiniReview indicates that multifaceted pharmacist-led interventions in a hospital setting may improve the quality of medication use and reduce hospital visits and length of stay, while no effect was seen on mortality, patient-reported outcome and cost-effectiveness.

The danish regions pediatric triage model has a limited ability to detect both critically ill children as well as children to be sent home without treatment - a study of diagnostic accuracy

BackgroundThe Danish Regions Pediatric Triage model (DRPT) was introduced in 2012 and subsequent implemented in most Danish acute pediatric departments. The aim was to evaluate the validity of DRPT as a screening tool to detect both the most serious acute conditions and the non-serious conditions in the acute referred patients in a pediatric department.MethodThe study was prospective observational, with follow-up on all children with acute referral to pediatric department from October to December 2015. The DRPT was evaluated by comparison to a predefined reference standard and to the actual clinical outcomes: critically ill children and children returned to home without any treatment. The sensitivity, specificity, positive predictive value, negative predictive value, accuracy and likelihood for positive and negative test were calculated.ResultsFive hundred fifty children were included. The DRPT categorized 7% very urgent, 28% urgent, 29% standard and 36% non-urgent. The DRPT was equal to the reference standard in 31% of the children (CI: 27-35%). DRPT undertriaged 55% of the children (CI: 51-59%) and overtriaged 14% of the children (CI: 11-17%). For the most urgent patients the sensitivity of DRPT was 31% (CI: 20-48%) compared to the reference standard and 20% (CI: 7-41) for critically ill. For children with non-urgent conditions the specificity of DRPT was 66% (CI: 62-71%) compared to the reference standard and 68% (CI: 62-75%) for the children who went home with no treatment. In none of the analyses, the likelihood ratio of the negative test was less than 0.7 and the positive likelihood ratio only reached more than 5 in one of the analyses.DiscussionThis study is the first to evaluate the DRPT triage system. From the very limited validity studies of other well-established triage systems, it is difficult to judge whether the DRPT performs better or worse than the alternatives. The DRPT errs to the undertriage side. If the sensitivity is low, a number of the sickest children are undetected and this is a matter of concern.ConclusionThe DRPT is a triage tool with limited ability to detect the critically ill children as well as the children who can be returned to home without any treatment.Trial registrationNot relevant

Do prehospital providers and emergency nurses agree on triage assignment?: an efficacy study

Objectives The aim of this study was to investigate the agreement on triage level between prehospital providers and emergency department (ED) nurses in clinical practice when using the same triage system. The objectives were as follows: (a) What is the agreement of triage between prehospital providers and ED nurses, when using Danish Emergency Process Triage (DEPT) correctly? (b) Which part of the triage process yields the highest agreement regarding the final triage? Methods The study was a prospective and observational efficacy study. Patients transported to the ED by ambulances were included. They were triaged by prehospital providers while being transported by ambulance to the ED, and by ED nurses upon arrival. Triage was done using the DEPT – a five-level triage system based on vital signs and a presenting complaint algorithm. An agreement analysis was performed. Results DEPT was used correctly by both professions in 292 patients. In 182 (62%) patients the prehospital providers and the ED nurses agreed on the same triage level. This equals to &kgr;=0.47 [95% confidence interval (CI): 0.41–0.56]. When considering the triage based on vital signs the agreement was 72% (&kgr;=0.46; 95% CI: 0.41–0.47), and based on presenting complaint the agreement was 46% (&kgr;=0.41; 95% CI: 0.37–0.44). Conclusion There was a moderate interrater agreement on triage assignment between ED nurses and prehospital providers. They agreed on final triage more often if they agreed on triage based on vital signs rather than presenting complaints.