Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Machine learning based outcome prediction in stroke patients with MCA‐M1 occlusions and early thrombectomy

Background Clinical outcome varies substantially between individuals with large vessel occlusion (LVO) stroke. A small infarct core and large mismatch were found to be associated with good recovery. We investigated if those imaging variables improve individual prediction of functional outcome after early (< 6h) endovascular treatment (EVT) in LVO stroke. Methods We included 222 patients with acute ischemic stroke due to middle cerebral artery (MCA)‐M1 occlusion who received EVT. As predictors, we used clinical variables and region of interest (ROI) based magnetic resonance imaging (MRI) features. We developed different machine learning models and quantified their prediction performance by the area under the curve (AUC) of receiver operator characteristics (ROC) curves and the Brier score. Results Successful recanalization rate was 78%, with 54% patients having a favorable outcome (modified Rankin scale, mRS 0‐2). Small infarct core was associated with favorable functional outcome. Outcome prediction improved only slightly when imaging was added to patient variables. Age was the driving factor, with a sharp decrease of likelihood for favorable functional outcome beyond 78 years of age. Conclusions In patients with MCA‐M1 occlusion strokes referred to EVT within 6 hours of symptom onset, infarct core volume was associated with outcome. However, ROI based imaging parameters led to no significant improvement in outcome prediction on individual patient level when added to a set of clinical predictors. Our study is in concordance with the current practice, where mismatch imaging or collateral readouts are not recommended for excluding patients with MCA‐M1 occlusion for early EVT

Early changes in morphology, bone mineral density and matrix composition of vertebrae lead to disc degeneration in aged collagen IX -/- mice

Collagen IX (Col IX) is an important component of the cartilage extracellular matrix and has been associated with degenerative cartilage disorders and chondrodysplasias in humans. Further, polymorphisms in Col IX are known risk factors for the development of early intervertebral disc (IVD) degeneration. To understand the role of Col IX in the pathogenesis of IVD disorders, the spine of newborn and older Col IX deficient mice was systematically analyzed and compared to C57BL/6N controls. Morphology and bone parameters of the spine from newborn, 6 and 10 months old animals were investigated using mu CT measurements. Histological staining was used to evaluate tissue structure and degree of degeneration. Localization and expression of extracellular matrix proteins was analyzed in depth by immunofluorescence staining, immunoblotting, RT-PCR and in situ hybridization. High resolution imaging and stiffness measurements were performed by atomic force microscopy (AFM). Vertebral bodies of newborn Col IX-deficient mice were smaller and showed an increased mineral density compared to wild type animals. At birth, lack of Col IX led to a disrupted cellular organization in the cartilaginous endplate and a smaller nucleus pulposus of the IVD. Expression levels and localization of other extracellular matrix proteins were strongly altered accompanied by a softening of cartilaginous tissues. In older animals, absence of Col IX caused earlier and more pronounced disc degeneration with annular fissures. The absence of Col IX induces early developmental, structural and biomechanical alterations in both vertebral body and intervertebral disc which eventually cause severe degenerative changes in the aging spine. (C) 2015 Elsevier B.V. All rights reserved

Individual hiPSCs varied in their susceptibility to allogeneic and autologous NK cells and NK cells of individual donors varied in their activity against allogeneic and autologous hiPSCs.

<p>(A) A summary of means of specific lysis and the SEM of D1-iPSC4 cells by allogeneic (allo) and autologous (auto) NK cells is shown. The numbers of individual experiments (n) are indicated in the figure. (B) A summary of means of specific lysis and the SEM of the autologous hiPSC line D1-iPSC4 and the two allogeneic hiPSC lines (D2-iPSC1, D3-iPSC3) by NK cells of donor 1 is shown. (C) A summary of means of specific lysis and the SEM of D2-iPSC1 cells by allogeneic (allo) and autologous (auto) NK cells is shown. (D) A summary of means of specific lysis and the SEM of the autologous hiPSC line D2-iPSC1 and the two allogeneic hiPSC lines (D1-iPSC4, D3-iPSC3) by NK cells of donor 2 is shown. (E) A summary of means of specific lysis and the SEM of D3-iPSC3 by allogeneic (allo) and autologous (auto) NK cells is shown. (F) A summary of means of specific lysis and the SEM of the autologous hiPSC line D3-iPSC3 and the two allogeneic hiPSC lines (D1-iPSC4, D2-iPSC1) by NK cells of donor 3 is shown.</p

Gene expression analysis of hiPSC lines indicated mostly low mRNA expression of activating and inhibitory NK receptor ligands.

<p>(A) The expression of the indicated genes was tested by qPCR in PBMCs, K562 cells and three hiPSC lines (D1-iPSC4, D2-iPSC1, and D3-iPSC3). For the hiPSC lines and K562 cells means ± SEM of Δct values (ct target gene [tg] minus ct housekeeping gene [hkg]) of three biological replicates are shown. Negative values indicated a higher expression of the target gene than the housekeeping gene. Therefore, an inverted scale is shown. At the left side, genes encoding for ligands of activating NK receptors and <i>ICAM1</i> are shown. In the middle part classical and non-classical HLA class I genes, <i>B2M</i>, and the HLA class II gene <i>DRA</i> are shown. In the right part, genes involved in antigen processing in the HLA class I pathway are grouped. Genes not expressed in the iPSC lines (ct > 30) are marked by Ø. (B) The gene expression in the iPSC lines is shown as relative expression compared to PBMCs. (C) The gene expression in the hiPSC lines is shown as relative expression compared to K562 cells.</p

Human iPSC lines were killed by purified and IL-2-activated NK cells of various donors but allogeneic effector cells were more efficient than autologous NK cells.

<p>(A) NK cells were stimulated for four days with IL-2 (200 U/ml) and used as effector cells against the reference target cell line K562 in ⁵¹Cr-release assays. Each individual test was done in triplicates. The means of specific lysis and the standard error of the mean (SEM) at different effector:target (E:T) ratios (16:1 to 0.25:1) are shown to summarize these experiments. The numbers of individual experiments (n) are indicated in the figure. (B) A summary of means of specific lysis and the SEM of K562 and three hiPSC lines by IL-2-activated NK cells from five donors (1 to 5) is shown. (C) A summary of means of specific lysis and the SEM of the three hiPSC lines (D1-iPSC4, D2-iPSC1, D3-iPSC3) by NK cells of five different donors is shown. (D) A summary of means of specific lysis and the SEM of the three hiPSC lines (D1-iPSC4, D2-iPSC1, D3-iPSC3) by allogeneic (allo) and autologous (auto) NK cells is shown.</p

NK cells degranulating in response to hiPSC lines are enriched for several NK cell receptors.

<p>(A) The degranulation of IL-2-activated NK cells of donor 7 in response to D6-iPSC2 cells is shown. Without contact to hiPSCs only 2.9% of the NK cells expressed the degranulation marker CD107a. After co-culture with target cells for 2 h 25.2% of the CD56⁺ NK cells expressed CD107a at the plasma membrane. 46.7% of the CD107a^- NK cells were KIR positive. Among the CD107a⁺ NK cells 53.0% were KIR positive. The KIR staining was performed with a mixture of all anti-KIR mAbs indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125544#pone.0125544.t001" target="_blank">Table 1</a> to cover all KIR molecules. (B) A summary of means and the SEM of CD107a⁺ NK cells of donors 4, 5, and 7 after exposure to three hiPSC lines (D1-iPSC4, D2-iPSC1, D6-iPSC2) and K562 cells is shown (n = 3). (C) In the left panel a summary of means and the SEM of NKG2D⁺, NKG2A⁺, DNAM-1⁺, and KIR⁺ cells among all NK cells exposed to the hiPSCs as well as CD107a^- and CD107a⁺ NK cells is shown. In the right panel a summary of means and the SEM of the MFI of NKG2D, NKG2A, DNAM-1, and KIR on all NK cells exposed to the hiPSCs as well as CD107a^- and CD107a⁺ NK cells is shown. Significant differences between CD107a^- and CD107a⁺ NK cells are indicated (n = 26, *** P<0.001, ** P<0.01, <i>t</i>-test after Bonferroni-Holm correction).</p

Flow cytometric analysis of ligands for activating and inhibitory NK receptors on D1-iPSC4 cells.

<p>Representative histograms of propidium iodide negative D1-iPSC4 cells are shown after staining with mAbs for the indicated molecules or with recombinant receptor molecules (NKG2D, NKp30, NKp44, and NKp46) for the respective ligands. Staining with the respective primary reagent is shown in red and with the secondary Ab only in black. The percentages of specifically stained cells (using the marker shown) and the specific MFI were calculated and are given in the figure.</p

Killing of hiPSC lines is in general dependent on DNAM-1 and for D6-iPSC2 cells also on HLA class I molecules.

<p>(A) A summary of means of specific lysis and the SEM of three hiPSC lines (D1-iPSC4, D2-iPSC1, D6-iPSC2) by IL-2-activated NK cells from the donors 4, 5, and 7 in ⁵¹Cr-release assays is shown after incubation with an isotype control (IgG₁) or blocking mAbs against NKG2D (aNKG2D), DNAM-1 (aDNAM-1), or ICAM-1 (aICAM-1) at a concentration of 10 μg/ml. The significance of differences was calculated by ANOVA and is indicated in the figure. The numbers of individual experiments (n) are indicated in the figure. (B) A summary of means of specific lysis and the SEM of three hiPSC lines (D1-iPSC4, D2-iPSC1, D6-iPSC2) by IL-2-activated NK cells from the donors 4, 5 and 7 is shown after incubation with the W6/32 HL mAb that binds to HLA class I molecules or as control the non-binding variant W6/32 HK at a concentration of 10 μg/ml. (C, D) The data for the three hiPSC lines are shown grouped according to the three NK cell donors. (E, F) The data are shown grouped according to the three hiPSC lines and K562 cells. Significant differences were calculated by 2-way-ANOVA adjusted either for the NK cell donors or the hiPSC lines and are indicated in the figure (** P<0.01, *P<0.05).</p