BackgroundCollagen deposition and an altered matrix metalloproteinase (MMP) expression profile are hallmarks of fibrosis. Type IV collagen is the most abundant structural basement membrane component of tissue, which increases 14-fold during fibrogenesis in the liver. Proteolytic degradation of collagens by proteases produces small fragments, so-called neoepitopes, which are released systemically. Technologies investigating MMP-generated fragments of collagens may provide more useful information than traditional serological assays that crudely measure total protein. In the present study, we developed an ELISA for the quantification of a neoepitope generated by MMP degradation of type IV collagen and evaluated the association of this neoepitope with liver fibrosis in two animal models.MethodsType IV collagen was degraded in vitro by a variety of proteases. Mass spectrometric analysis revealed more than 200 different degradation fragments. A specific peptide sequence, 1438'GTPSVDHGFL'1447 (CO4-MMP), in the α1 chain of type IV collagen generated by MMP-9 was selected for ELISA development. ELISA was used to determine serum levels of the CO4-MMP neoepitope in two rat models of liver fibrosis: inhalation of carbon tetrachloride (CCl4) and bile duct ligation (BDL). The levels were correlated to histological findings using Sirius red staining.ResultsA technically robust assay was produced that is specific to the type IV degradation fragment, GTPSVDHGFL. CO4-MMP serum levels increased significantly in all BDL groups compared to baseline, with a maximum increase of 248% seen two weeks after BDL. There were no changes in CO4-MMP levels in sham-operated rats. In the CCl4 model, levels of CO4-MMP were significantly elevated at weeks 12, 16 and 20 compared to baseline levels, with a maximum increase of 88% after 20 weeks. CO4-MMP levels correlated to Sirius red staining results.ConclusionThis ELISA is the first assay developed for assessment of proteolytic degraded type IV collagen, which, by enabling quantification of basement membrane degradation, could be relevant in investigating various fibrogenic pathologies. The CO4-MMP degradation fragment was highly associated with liver fibrosis in the two animal models studied.

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PurposeAbstractTo determine the validity of the Australian clinical prediction tool Criteria for Screening and Triaging to Appropriate aLternative care (CRISTAL) based on objective clinical criteria to accurately identify risk of death within 3 months of admission among older patients.MethodsProspective study of ≥ 65 year-olds presenting at emergency departments in five Australian (Aus) and four Danish (DK) hospitals. Logistic regression analysis was used to model factors for death prediction; Sensitivity, specificity, area under the ROC curve and calibration with bootstrapping techniques were used to describe predictive accuracy.Results2493 patients, with median age 78–80 years (DK–Aus). The deceased had significantly higher mean CriSTAL with Australian mean of 8.1 (95% CI 7.7–8.6 vs. 5.8 95% CI 5.6–5.9) and Danish mean 7.1 (95% CI 6.6–7.5 vs. 5.5 95% CI 5.4–5.6). The model with Fried Frailty score was optimal for the Australian cohort but prediction with the Clinical Frailty Scale (CFS) was also good (AUROC 0.825 and 0.81, respectively). Values for the Danish cohort were AUROC 0.764 with Fried and 0.794 using CFS. The most significant independent predictors of short-term death in both cohorts were advanced malignancy, frailty, male gender and advanced age. CriSTAL’s accuracy was only modest for in-hospital death prediction in either setting.ConclusionsThe modified CriSTAL tool (with CFS instead of Fried’s frailty instrument) has good discriminant power to improve prognostic certainty of short-term mortality for ED physicians in both health systems. This shows promise in enhancing clinician’s confidence in initiating earlier end-of-life discussions.Electronic supplementary materialThe online version of this article (10.1007/s41999-018-0123-6) contains supplementary material, which is available to authorized users.

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.

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.

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.

ObjectivesThe aim of this study was to describe the carrier prevalence and demographic variation of four different multiresistant bacteria (MRB) among acute patients in Danish emergency departments (EDs): methicillin-resistantStaphylococcus aureus(MRSA), carbapenemase-producing enterobacteria (CPE), extended-spectrum beta-lactamase-producing enterobacteria (ESBL) and vancomycin-resistant enterococci (VRE), and to analyse the association of MRB carriage to a range of potential risk factors.DesignMulticentre descriptive and analytic cross-sectional survey.SettingEight EDs and four clinical microbiology departments in Denmark.ParticipantsAdults visiting the ED.Main outcome measuresSwabs from nose, throat and rectum were collected and analysed for MRSA, ESBL, VRE and CPE. The primary outcome was the prevalence of MRB carriage, and secondary outcomes relation to risk factors among ED patients.ResultsWe included 5117 patients in the study. Median age was 68 years (54–77) and gender was equally distributed. In total, 266 (5.2%, 95% CI 4.6 to 5.8) were colonised with at least one MRB. No significant difference was observed between male and female patients, between age groups and between university and regional hospitals. Only 5 of the 266 patients with MRB were colonised with two of the included bacteria and none with more than two. CPE prevalence was 0.1% (95% CI 0.0 to 0.2), MRSA prevalence was 0.3% (95% CI 0.2 to 0.5), VRE prevalence was 0.4% (95% CI 0.3 to 0.6) and ESBL prevalence was 4.5% (95% CI 3.9 to 5.1). Risk factors for MRB carriage were previous antibiotic treatment, previous hospital stay, having chronic respiratory infections, use of urinary catheter and travel to Asia, Oceania or Africa.ConclusionEvery 20th patient arriving to a Danish ED brings MRB to the hospital. ESBL is the most common MRB in the ED. The main risk factors for MRB carriage are recent antibiotic use and travel abroad.Trial registration numberNCT03352167;Post-results.

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.