Dual bronchodilators in Bronchiectasis study (DIBS): protocol for a pragmatic, multicentre, placebo-controlled, three-arm, double-blinded, randomised controlled trial studying bronchodilators in preventing exacerbations of bronchiectasis.

INTRODUCTION: Bronchiectasis is a long-term lung condition, with dilated bronchi, chronic inflammation, chronic infection and acute exacerbations. Recurrent exacerbations are associated with poorer clinical outcomes such as increased severity of lung disease, further exacerbations, hospitalisations, reduced quality of life and increased risk of death. Despite an increasing prevalence of bronchiectasis, there is a critical lack of high-quality studies into the disease and no treatments specifically approved for its treatment. This trial aims to establish whether inhaled dual bronchodilators (long acting beta agonist (LABA) and long acting muscarinic antagonist (LAMA)) taken as either a stand-alone therapy or in combination with inhaled corticosteroid (ICS) reduce the number of exacerbations of bronchiectasis requiring treatment with antibiotics during a 12 month treatment period. METHODS: This is a multicentre, pragmatic, double-blind, randomised controlled trial, incorporating an internal pilot and embedded economic evaluation. 600 adult patients (≥18 years) with CT confirmed bronchiectasis will be recruited and randomised to either inhaled dual therapy (LABA+LAMA), triple therapy (LABA+LAMA+ICS) or matched placebo, in a 2:2:1 ratio (respectively). The primary outcome is the number of protocol defined exacerbations requiring treatment with antibiotics during the 12 month treatment period. ETHICS AND DISSEMINATION: Favourable ethical opinion was received from the North East-Newcastle and North Tyneside 2 Research Ethics Committee (reference: 21/NE/0020). Results will be disseminated in peer-reviewed publications, at national and international conferences, in the NIHR Health Technology Assessments journal and to participants and the public (using lay language). TRIAL REGISTRATION NUMBER: ISRCTN15988757.

Acipimox in Mitochondrial Myopathy (AIMM): study protocol for a randomised, double-blinded, placebo-controlled, adaptive design trial of the efficacy of acipimox in adult patients with mitochondrial myopathy.

BACKGROUND: Mitochondrial disease is a heterogenous group of rare, complex neurometabolic disorders. Despite their individual rarity, collectively mitochondrial diseases represent the most common cause of inherited metabolic disorders in the UK; they affect 1 in every 4300 individuals, up to 15,000 adults (and a similar number of children) in the UK. Mitochondrial disease manifests multisystem and isolated organ involvement, commonly affecting those tissues with high energy demands, such as skeletal muscle. Myopathy manifesting as fatigue, muscle weakness and exercise intolerance is common and debilitating in patients with mitochondrial disease. Currently, there are no effective licensed treatments and consequently, there is an urgent clinical need to find an effective drug therapy. AIM: To investigate the efficacy of 12-week treatment with acipimox on the adenosine triphosphate (ATP) content of skeletal muscle in patients with mitochondrial disease and myopathy. METHODS: AIMM is a single-centre, double blind, placebo-controlled, adaptive designed trial, evaluating the efficacy of 12 weeks' administration of acipimox on skeletal muscle ATP content in patients with mitochondrial myopathy. Eligible patients will receive the trial investigational medicinal product (IMP), either acipimox or matched placebo. Participants will also be prescribed low dose aspirin as a non-investigational medical product (nIMP) in order to protect the blinding of the treatment assignment. Eighty to 120 participants will be recruited as required, with an interim analysis for sample size re-estimation and futility assessment being undertaken once the primary outcome for 50 participants has been obtained. Randomisation will be on a 1:1 basis, stratified by Fatigue Impact Scale (FIS) (dichotomised as < 40, ≥ 40). Participants will take part in the trial for up to 20 weeks, from screening visits through to follow-up at 16 weeks post randomisation. The primary outcome of change in ATP content in skeletal muscle and secondary outcomes relating to quality of life, perceived fatigue, disease burden, limb function, balance and walking, skeletal muscle analysis and symptom-limited cardiopulmonary fitness (optional) will be assessed between baseline and 12 weeks. DISCUSSION: The AIMM trial will investigate the effect of acipimox on modulating muscle ATP content and whether it can be repurposed as a new treatment for mitochondrial disease with myopathy. TRIAL REGISTRATION: EudraCT2018-002721-29 . Registered on 24 December 2018, ISRCTN 12895613. Registered on 03 January 2019, https://www.isrctn.com/search?q=aimm.

Evaluation of stroke thrombectomy including patients where IV thrombolysis is contraindicated or has failed: a randomized trial of two novel thrombectomy devices.

BACKGROUND: Study was a PROBE design phase II randomized controlled trial (RCT). We assessed trial feasibility and technical efficacy and safety of two novel thrombectomy devices - ERIC (a retriever device) and SOFIA (a distal access catheter) - used alone or in combination depending on operator preference. METHODS: Four UK neuroscience centers enrolled adults with proximal large artery occlusion (LAO) stroke on imaging where arterial puncture was achievable within 5.5 hours (8.5 hours for posterior circulation) of symptom onset; National Institutes of Health Stroke Scale (NIHSS) ≥6 with limited ischemic change on CT imaging. Randomization was 2:1 into intervention arm (ERIC and/or SOFIA). Patients and core lab were blinded to allocation. Primary outcome was independent core lab adjudication of reperfusion (modified Thrombolysis in Cerebral Infarction (mTICI) scale). Secondary outcomes were modified Rankin score (mRS) at 90 and 365 days (independence and shift analysis), 30-day mortality, symptomatic intracranial hemorrhage (sICH), procedural complications and NIHSS change. RESULTS: Sixty-six patients were enrolled. TICI 2B/3 reperfusion was achieved in 72% in intervention compared with 90% in control arm on intention to treat (ITT) analysis (P=0.2) and 78% compared with 86% on per protocol analysis (P=0.7). Functional independence at 90 days was 40% (intervention) compared with 43% (control) on ITT analysis (P=1.0). sICH rates were low at 0% and 5%, respectively (P=0.3). The 30-day mortality was 9% intervention compared with 14% control (P=0.7). CONCLUSIONS: Study indicated feasibility of a phase II RCT trial approach for assessing new thrombectomy devices. In a broad LAO stroke population ERIC and SOFIA were not statistically different from control devices. Larger trials are needed.

Lipophilic statins prevent matrix metalloproteinase-mediated cartilage collagen breakdown by inhibiting protein geranylgeranylation.

OBJECTIVE: To investigate if statins prevent cartilage degradation and the production of collagenases and gelatinases in bovine nasal and human articular cartilage after proinflammatory cytokine stimulation. METHODS: In a cartilage degradation model, the effects of several statins were assessed by measuring proteoglycan degradation and collagen degradation, while collagenolytic and gelatinolytic activity in culture supernatants were determined by collagen bioassay and gelatin zymography. The production of matrix metalloproteinases (MMPs) in cartilage and chondrocytes were analysed by real-time reverse transcriptase PCR and immunoassay. Cytokine-induced signalling pathway activation was studied by immunoblotting. RESULTS: Simvastatin and mevastatin significantly inhibited interleukin 1 (IL-1)+oncostatin M (OSM)-induced collagen degradation; this was accompanied with a marked decrease in collagenase and gelatinase activity from bovine nasal cartilage. The cholesterol pathway intermediate mevalonic acid reversed the simvastatin-mediated protection of cartilage degradation, and the expression and production of collagenase (MMP-1 and MMP-13) and gelatinase (MMP-2 and MMP-9). Statins also significantly decreased MMP-1 and MMP-13 expression in human articular cartilage and chondrocytes stimulated with IL-1+OSM, and blocked the activation of critical proinflammatory signalling pathways required for MMP expression. The loss of the isoprenoid intermediate geranylgeranyl pyrophosphate due to statin treatment accounted for the inhibition of MMP expression and signalling pathway activation. CONCLUSIONS: This study shows, for the first time, that lipophilic statins are able to block cartilage collagen breakdown induced by proinflammatory cytokines, by downregulating key cartilage-degrading enzymes. This demonstrates a possible therapeutic role for statins in acting as anti-inflammatory agents and in protecting cartilage from damage in joint diseases.

Effect of an Enhanced Paramedic Acute Stroke Treatment Assessment on Thrombolysis Delivery During Emergency Stroke Care: A Cluster Randomized Clinical Trial.

Importance: Rapid thrombolysis treatment for acute ischemic stroke reduces disability among patients who are carefully selected, but service delivery is challenging. Objective: To determine whether an enhanced Paramedic Acute Stroke Treatment Assessment (PASTA) intervention increased hospital thrombolysis rates. Design, Setting, and Participants: This multicenter, cluster randomized clinical trial took place between December 2015 and July 2018 in 3 ambulance services and 15 hospitals. Clusters were paramedics based within ambulance stations prerandomized to PASTA or standard care. Patients attended by study paramedics were enrolled after admission if a hospital specialist confirmed a stroke and paramedic assessment started within 4 hours of onset. Allocation to PASTA or standard care reflected the attending paramedic's randomization status. Interventions: The PASTA intervention included additional prehospital information collection, a structured hospital handover, practical assistance up to 15 minutes after handover, a predeparture care checklist, and clinician feedback. Standard care reflected national guidelines. Main Outcomes and Measures: Primary outcome was the proportion of patients receiving thrombolysis. Secondary outcomes included time intervals and day 90 health (with poor status defined as a modified Rankin Score >2, to represent dependency or death). Results: A total of 11 478 patients were screened following ambulance transportation; 1391 were eligible and approached, but 177 did not consent. Of 1214 patients enrolled (mean [SD] age, 74.7 [13.2] years; 590 women [48.6%]), 500 were assessed by 242 paramedics trained in the PASTA intervention and 714 were assessed by 355 paramedics continuing with standard care. The paramedics trained in the PASTA intervention took a mean of 13.4 (95% CI, 9.4-17.4) minutes longer (P < .001) to complete patient care episodes. There was less thrombolysis among the patients in the PASTA group, but this was not significant (PASTA group, 197 of 500 patients [39.4%] vs the standard care group, 319 of 714 patients [44.7%]; adjusted odds ratio, 0.81 [95% CI, 0.61-1.08]; P = .15). Time from a paramedic on scene to thrombolysis was a mean of 8.5 minutes longer in the PASTA group (98.1 [37.6] minutes) vs the standard care group (89.4 [31.1] minutes; P = .01). Poor health outcomes did not differ significantly but occurred less often among patients in the PASTA group (313 of 489 patients [64.0%]) vs the standard care group (461 of 690 patients [66.8%]; adjusted odds ratio, 0.86 [95% CI, 0.60-1.20]; P = .39). Conclusions and Relevance: An enhanced paramedic assessment did not facilitate thrombolysis delivery. The unexpected combination of thrombolysis and health outcomes suggests possible alternative influences on treatment decisions by the intervention, requiring further evaluation. Trial Registration: ISRCTN Registry Identifier: ISRCTN12418919.

Sulfasalazine blocks the release of proteoglycan and collagen from cytokine stimulated cartilage and down-regulates metalloproteinases.

OBJECTIVE: To investigate the effect of SSZ on the release of GAG and collagen fragments from bovine nasal cartilage and MMP and ADAMTS (a disintegrin and metalloproteinase domain with thrombospondin motifs) proteinases from human articular chondrocytes (HACs) stimulated with IL-1alpha and oncostatin M (OSM). METHODS: SSZ was added to bovine nasal explant cultures stimulated to resorb with IL-1alpha and OSM, and the release of GAG and collagen has been determined. Collagenolytic activity was measured using the radio-labelled collagen bioassay. HACs were treated with IL-1alpha and OSM with and without SSZ, and MMP-1 and -13 and ADAMTS-4 and -5 were measured for protein and gene expression by ELISA and RT-PCR, respectively. RESULTS: SSZ blocked GAG and collagen fragment release from bovine cartilage, and reduced active and total collagenase activity in a dose-dependent manner. SSZ transcriptionally blocked MMP-1, -13 and ADAMTS-4, and reduced the protein levels of MMP-1 and -13 in a dose-dependent manner following stimulation of HACs with IL-1alpha and OSM. CONCLUSION: This study shows for the first time that SSZ blocks release of proteoglycan and collagen fragments from resorbing cartilage and lowers the levels of proteoglycan and collagen-degrading enzymes. These results indicate that in addition to acting as an anti-inflammatory agent, SSZ may have a therapeutic role in protecting cartilage from damage in OA.

A novel paradigm for dendritic cells as effectors of cartilage destruction.

OBJECTIVE: Dendritic cells (DCs) are enriched in RA synovium and have been implicated in the pathogenesis of RA primarily through their ability to present autoantigen and activate T cells. However, whether DCs play an effector role in cartilage destruction is unknown. The aim of this study was to investigate whether DCs can induce collagen release from cartilage and the mechanism involved. METHODS: Human monocyte-derived DCs (mDCs) were activated with CD40 ligand (CD40L) to mimic DC-T-cell interaction, and supernatants were incubated with cartilage explants. Hydroxyproline was assessed as a measure of collagen release and collagenolytic activity was measured by a bioassay using tritiated collagen. TNF-alpha in DC supernatants was measured by specific ELISA. RESULTS: Supernatants from CD40L-activated mDCs, but not unstimulated mDCs, strongly induced the destruction of cartilage collagen. mDC supernatants did not contain collagenases but did induce collagenolytic activity in cartilage explants. Neutralization of TNF-alpha in mDC supernatants completely abolished collagenolysis. CONCLUSIONS: This study shows that mDCs, upon CD40-ligation, induce cartilage collagen degradation through an indirect mechanism via the production of TNF-alpha. Our data suggest a potential important role for mDC-derived TNF-alpha in RA, which is in line with the previously reported observations that DCs are a major source of TNF-alpha in early autoimmune lesions and that anti-TNF-alpha therapeutics effectively suppress joint damage in RA patients. We propose that DCs can act as effectors in cartilage destruction, adding a new aspect to the functional role of DCs in RA pathogenesis.

Paramedic Acute Stroke Treatment Assessment (PASTA): study protocol for a randomised controlled trial.

BACKGROUND: Despite evidence from clinical trials that intravenous (IV) thrombolysis is a cost-effective treatment for selected acute ischaemic stroke patients, there remain large variations in the rate of IV thrombolysis delivery between stroke services. This study is evaluating whether an enhanced care pathway delivered by paramedics (the Paramedic Acute Stroke Treatment Assessment (PASTA)) could increase the number of patients who receive IV thrombolysis treatment. METHODS: Study design: Cluster randomised trial with economic analysis and parallel process evaluation. SETTING: National Health Service ambulance services, emergency departments and hyper-acute stroke units within three geographical regions of England and Wales. Randomisation: Ambulance stations within each region are the units of randomisation. According to station allocation, paramedics based at a station deliver the PASTA pathway (intervention) or continue with standard stroke care (control). Study intervention: The PASTA pathway includes structured pre-hospital information collection, prompted pre-notification, structured handover of information in hospital and assistance with simple tasks during the initial hospital assessment. Study-trained intervention group paramedics deliver this pathway to adults within 4 h of suspected stroke onset. Study control: Standard stroke care according to national and local guidelines for the pre-hospital and hospital assessment of suspected stroke. PARTICIPANTS: Participants enrolled in the study are adults with confirmed stroke who were assessed by a study paramedic within 4 h of symptom onset. PRIMARY OUTCOME: Proportion of participants receiving IV thrombolysis. SAMPLE SIZE: 1297 participants provide 90% power to detect a 10% difference in the proportion of patients receiving IV thrombolysis. DISCUSSION: The results from this trial will determine whether an enhanced care pathway delivered by paramedics can increase thrombolysis delivery rates. TRIAL REGISTRATION: ISRCTN registry, ISRCTN12418919 . Registered on 5 November 2015.

Baseline serum MMP-3 levels in patients with Rheumatoid Arthritis are still independently predictive of radiographic progression in a longitudinal observational cohort at 8 years follow up.

INTRODUCTION: At present, there is no reliable tool for predicting disease outcome in patients with rheumatoid arthritis (RA). We previously demonstrated an association between specific baseline biomarkers/clinical measures including matrix metalloproteinase-3 (MMP-3) and 2-year radiographic progression in patients with RA. This study further evaluates the predictive capability of these baseline variables with outcome extended over 8-years. METHODS: Fifty-eight of the original cohort (n = 118) had radiographic progression from baseline to mean 8.2-years determined using the van der Heijde modified Sharp method. The contribution of each predictor variable towards radiographic progression was assessed with univariate and multivariate analyses. RESULTS: Traditional factors (including erythrocyte sedimentation rate, C-reactive protein, anti-cyclic citrullinated peptide (anti-CCP), and rheumatoid factor) and biomarkers of tissue destruction (including MMP-3, C-telopeptide of type II collagen, cartilage oligomeric matrix protein, and tissue inhibitor of metalloproteinase 1) measured at baseline were associated with radiographic progression at endpoint. Multivariate logistic regression identified anti-CCP seropositivity [OR 9.29, 95%CI: 2.29-37.64], baseline elevated MMP-3 [OR 8.25, 95%CI: 2.54-26.78] and baseline radiographic damage [OR 5.83, 95%CI: 1.88-18.10] as the strongest independent predictors of radiographic progression. A model incorporating these variables had a predictive accuracy of 0.87, assessed using the area under the receiver operating characteristic curve. CONCLUSION: In our cohort with onset of RA symptoms < 2-years, multivariate analysis identified anti-CCP status and baseline MMP-3 as the strongest independent predictors of radiographic disease outcome at 8.2-years. This finding suggests determination of baseline MMP-3, in conjunction with traditional serologic markers, may provide additional prognostic information for patients with RA. Furthermore, these findings highlight the importance of continued research into a broad range of biomarkers as potential predictors of joint damage.

Assay of matrix metalloproteinases against matrix substrates.

The assays described allow the activity of members of the matrix metalloproteinase (MMP) family that degrade collagen, gelatin and casein substrates to be measured. The protocols described include the preparation of radiolabeled substrates, methods for the separation of degraded product from undegraded substrate, and methods for the activation of MMPs. The advantages and disadvantages of these methods are discussed in relation to immunoassays that measure the amount of individual MMPs.

A novel in vitro model to investigate behavior of articular chondrocytes in osteoarthritis.

OBJECTIVE: To investigate in vivo simulation of the microenvironment in which osteoarthritis (OA) chondrocytes are cultured in vitro. METHODS: Human articular chondrocytes were cultured under normoxic and hypoxic conditions. Cells were cultured on standard culture plastic or a porous polyHEMA surface that closely resembles the in vivo cartilage microarchitecture. Morphological changes to the cells were demonstrated by fluorescent staining with DAPI and vinculin. Proteoglycan and type II collagen protein levels were assessed using established techniques. Matrix metalloproteinase-1 (MMP-1) production was assessed by ELISA. The gene expression of type II collagen and SOX9 was measured using real-time polymerase chain reaction. RESULTS: Cells grown on culture plastic were seen to be flat and hexagonal. Cells cultured on the porous polyHEMA surface exhibited morphology in keeping with the in vivo microenvironment. Glycosaminoglycan release in hypoxia was high from cells cultured on standard culture plastic. Transcriptional expression of type II collagen was upregulated in hypoxia and by culture on the polyHEMA surface. Transcriptional expression of SOX9 in hypoxia was upregulated compared to normoxia; no significant effect was seen by varying the culture surface. Translational expression of type II collagen was upregulated at 20% oxygen on the polyHEMA surface compared to culture plastic and this was related to MMP-1 expression. CONCLUSION: Culture of chondrocytes in hypoxia and on a porous surface simulates the in vivo microenvironment and illustrates the molecular mechanisms of OA.

Synergistic collagenase expression and cartilage collagenolysis are phosphatidylinositol 3-kinase/Akt signaling-dependent.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway has emerged as a major regulator of cellular functions and has been implicated in several pathologies involving remodeling of extracellular matrix (ECM). The end stage of inflammatory joint diseases is characterized by excessive ECM catabolism, and in this study we assess the role of PI3K signaling in the induction of collagenolytic matrix metalloproteinases (MMPs) in human chondrocytes. We used the most potent cytokine stimulus reported to promote cartilage ECM catabolism, namely interleukin-1 (IL-1) in combination with oncostatin M (OSM). Both OSM and IL-6 (in the presence of its soluble receptor), but not IL-1 nor leukemia inhibitory factor, induced Akt phosphorylation in human chondrocytes. Inhibition of PI3K signaling using LY294002 blocked IL-1+OSM-mediated Akt phosphorylation, induction of MMP-1 and MMP-13, and cartilage collagenolysis. To further explore the role of downstream substrates within the PI3K pathway, complementary use of small molecule inhibitors and specific small interfering RNAs demonstrated that the PI3K subunit p110alpha and Akt1 were required for MMP-1 mRNA induction. MMP-13 induction was also reduced by loss of function of these molecules and by a lack of p110delta, 3-phosphoinositide-dependent kinase-1 or Akt3. We therefore propose that the activities of specific elements of the PI3K signaling pathway, including Akt, are necessary for the synergistic induction of MMP-1 and MMP-13 and the cartilage breakdown stimulated by IL-1+OSM. Our data provide new insight into the mechanism of synergy between IL-1 and OSM and highlight new therapeutic targets for inflammatory joint diseases that aim to repress the expression of collagenases.

Histone deacetylase inhibitors modulate metalloproteinase gene expression in chondrocytes and block cartilage resorption.

Cartilage destruction in the arthritides is thought to be mediated by two main enzyme families: the matrix metalloproteinases (MMPs) are responsible for cartilage collagen breakdown, and enzymes from the ADAMTS (a disintegrin and metalloproteinase domain with thrombospondin motifs) family mediate cartilage aggrecan loss. Many genes subject to transcriptional control are regulated, at least in part, by modifications to chromatin, including acetylation of histones. The aim of this study was to examine the impact of histone deacetylase (HDAC) inhibitors on the expression of metalloproteinase genes in chondrocytes and to explore the potential of these inhibitors as chondroprotective agents. The effects of HDAC inhibitors on cartilage degradation were assessed using a bovine nasal cartilage explant assay. The expression and activity of metalloproteinases was measured using real-time RT-PCR, western blot, gelatin zymography, and collagenase activity assays using both SW1353 chondrosarcoma cells and primary human chondrocytes. The HDAC inhibitors trichostatin A and sodium butyrate potently inhibit cartilage degradation in an explant assay. These compounds decrease the level of collagenolytic enzymes in explant-conditioned culture medium and also the activation of these enzymes. In cell culture, these effects are explained by the ability of HDAC inhibitors to block the induction of key MMPs (e.g. MMP-1 and MMP-13) by proinflammatory cytokines at both the mRNA and protein levels. The induction of aggrecan-degrading enzymes (e.g. ADAMTS4, ADAMTS5, and ADAMTS9) is also inhibited at the mRNA level. HDAC inhibitors may therefore be novel chondroprotective therapeutic agents in arthritis by virtue of their ability to inhibit the expression of destructive metalloproteinases by chondrocytes.