Christian Medom Madsen

Molecular Characterization of Oxysterol Binding to the Epstein-Barr Virus-induced Gene 2 (GPR183)

Background: EBI2 was recently deorphanized as the first oxysterol-activated receptor, but the binding mode remains unknown. Results: Mutational analysis showed that substitution of Arg-87, Tyr-112, Tyr-116, and Tyr-260 abolished agonist binding and EBI2 activation. Conclusion: Oxysterol binding is dependent on residues in TM-II, -III, and -VI. Significance: This is the first study to examine the binding mode of these novel 7TM receptor agonists. Oxysterols are oxygenated cholesterol derivates that are emerging as a physiologically important group of molecules. Although they regulate a range of cellular processes, only few oxysterol-binding effector proteins have been identified, and the knowledge of their binding mode is limited. Recently, the family of G protein-coupled seven transmembrane-spanning receptors (7TM receptors) was added to this group. Specifically, the Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) was shown to be activated by several oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC). Nothing is known about the binding mode, however. Using mutational analysis, we identify here four key residues for 7α,25-OHC binding: Arg-87 in TM-II (position II:20/2.60), Tyr-112 and Tyr-116 (positions III:09/3.33 and III:13/3.37) in TM-III, and Tyr-260 in TM-VI (position VI:16/6.51). Substituting these residues with Ala and/or Phe results in a severe decrease in agonist binding and receptor activation. Docking simulations suggest that Tyr-116 interacts with the 3β-OH group in the agonist, Tyr-260 with the 7α-OH group, and Arg-87, either directly or indirectly, with the 25-OH group, although nearby residues likely also contribute. In addition, Tyr-112 is involved in 7α,25-OHC binding but via hydrophobic interactions. Finally, we show that II:20/2.60 constitutes an important residue for ligand binding in receptors carrying a positively charged residue at this position. This group is dominated by lipid- and nucleotide-activated receptors, here exemplified by the CysLTs, P2Y12, and P2Y14. In conclusion, we present the first molecular characterization of oxysterol binding to a 7TM receptor and identify position II:20/2.60 as a generally important residue for ligand binding in certain 7TM receptors.

Small molecule antagonism of oxysterol-induced Epstein-Barr virus induced gene 2 (EBI2) activation.

The Epstein–Barr virus induced gene 2 (EBI2) was recently identified as the first oxysterol‐activated 7TM receptor. EBI2 is essential for B cell trafficking within lymphoid tissues and thus the humoral immune response in general. Here we characterize the antagonism of the non‐peptide molecule GSK682753A, which blocks oxysterol‐induced G‐protein activation, β‐arrestin recruitment and B‐cell chemotaxis. We furthermore demonstrate that activation triggers pertussis toxin‐sensitive MAP kinase phosphorylation, which is also inhibited by GSK682753A. Thus, EBI2 signalling in B cells mediates key phenotypic functions via signalling pathways amenable to manipulation providing additional therapeutic options for inhibiting EBI2 activity.

Secondary hyperparathyroidism and mortality in hip fracture patients compared to a control group from general practice

INTRODUCTION Previously, little attention has been paid as to how disturbances in the parathyroid hormone (PTH)-calcium-vitamin D-axis, such as secondary hyperparathyroidism (SHPT), relate to mortality amongst hip fracture patients. This study aimed to (1) determine if SHPT is associated with mortality in this group of patients, (2) investigate the association between serum (s-) PTH, s-total calcium, s-25-hydroxyvitamin D (s-25(OH)D) and mortality and (3) determine the prevalence of SHPT amongst hip fracture patients and a control group. METHOD The study included 562 hip fracture patients (HF) (age ≥ 70 years) admitted to a Danish university hospital. The hip fracture patients were prospectively enrolled in a dedicated hip fracture database. Each hip fracture patient was exactly matched according to age and sex with two controls randomly chosen from a control population of 21,778 subjects who had s-PTH, s-total calcium and s-25(OH)D measured at the Copenhagen General Practitioners Laboratory after referral from their general practitioner. The control group (Con) thus consisted of 1124 subjects. RESULTS General 1-year mortality: Con-female 8.4%, Con-male 15.3%, HF-female 24.6%, HF-male 33.3%, p<0.0001 (log rank). SHPT AND RELATED 1-YEAR MORTALITY: Con-no SHPT 8.9%, Con-SHPT 16.8%, HF-no SHPT 22.7%, HF-SHPT 34.9%, p<0.0001 (log rank). The mortality rates were higher for controls with SHPT (OR 2.06, 95% CI: 1.32-3.23), hip fracture patients without SHPT (OR 3.00, 95% CI: 2.14-4.20) and hip fracture patients with SHPT (OR 5.46, 95% CI: 3.32-8.97) compared to the controls without SHPT. PREVALENCE OF SHPT: Con 16%, HF 20%, p=0.09 (Chi-square). CONCLUSIONS Our study clearly shows that SHPT is significantly associated with mortality in both hip fracture patients and the control group. In the multivariate Cox regression analysis, s-PTH and s-total calcium were both significantly associated with mortality, whereas s-25(OH)D was not associated with mortality in this analysis. Our study furthermore indicates that SHPT is almost equally prevalent amongst the hip fracture patients and the control group.

Orthogeriatric Service Reduces Mortality in Patients With Hip Fracture

Introduction: Orthogeriatric service has been shown to improve outcomes in patients with hip fracture. The purpose of this study is to evaluate the effect of orthogeriatrics at Bispebjerg University Hospital, Denmark. The primary outcome is mortality inhospital and after 1, 3, and 12 months for patients with hip fracture. The secondary outcome is mortality for home dwellers and nursing home inhabitants. Materials and Methods: This is a retrospective clinical cohort study with an historic control group including all patients with hip fracture admitted from 2007 to 2011. Patients with hip fracture are registered in a local database, and data are retrieved retrospectively using the Danish Civil Registration Number. Results: We included 993 patients in the intervention group and 989 patients in the control group. A univariate analysis showed only significantly decreased mortality inhospital 6.3% vs 3.1% (P = .009) after orthogeriatrics. However, when adjusting for age, gender, and American Society of Anaesthesiologists (ASA) score in a multivariate analysis, including all patients with hip fracture, we find significantly reduced mortality inhospital (odds ratio [OR] 0.35), after 30 [OR 0.66] and 90 days [OR 0.72] and 1 year [OR 0.79]). When using a univariate analysis for home-dwelling patients, we found significantly reduced mortality inhospital (8.3-2.0%, P < .0001), after 30 days (12.2-6.8%, P = .004) and 90 days (20.5-13.0%, P = .002). One-year mortality was not significant. Patients from nursing homes had no significant decreasing mortality at any point of time in the univariate analysis. Conclusion: We have shown significant decreases for inhospital, 30 day, 90 day, and 1-year mortality after implementation of orthogeriatric service at Bispebjerg Hospital when adjusting for age, gender, and ASA score. Future trials should include frail patients with other fracture types who can benefit from orthogeriatrics.

Low Levels of Hemoglobin at Admission Are Associated With Increased 30-Day Mortality in Patients With Hip Fracture

Introduction: Previous smaller studies suggest that anemia is a risk factor for mortality in patients with hip fracture. The purpose of this investigation was to assess the correlation between hemoglobin at admission with 30-day mortality following a hip fracture in a large-scale study. Patients and Methods: From January 1996 to December 2012, all patients with hip fracture (>60 years of age) admitted to Bispebjerg Hospital, Copenhagen, were identified from a local hip fracture database. We excluded conservatively treated patients and patients who died preoperatively. Results: Seven thousand four hundred twenty-one consecutive patients with hip fracture were identified. Of those 7319 had a hemoglobin measurement on admission and were thus eligible for further analysis. Mean hemoglobin for patients alive at 30 days was 7.6 (standard deviation [SD]: 1.0) and for deceased patients 7.4 (SD: 1.1), P < .0001. Mean age was 82.6 years (SD: 8.5), and 76.5% of the population were female (Nfemales = 5600). The 30-day mortality decreases for every increase in hemoglobin of 1.0 mmol/L in a univariate analysis (P < .0001). The hazard ratio (HR) with 95% confidence interval (CI) for 30-day mortality in patients with anemia (<7.3 mmol/L for females and <8.3 mmol/L for males; Nanemic = 3235) was 1.66 (CI: 1.43-1.91, P < .0001). Adjusting for age, type of fracture, gender, and comorbidities (Charlson score) slightly attenuated the risk estimate (HR: 1.21, CI: 1.03-1.41, P = .02). Conclusion: This study demonstrates increased 30-day mortality in patients with low hemoglobin at admission, even after adjusting for comorbidities.

Biased agonism and allosteric modulation of G protein‐coupled receptor 183 – a 7TM receptor also known as Epstein–Barr virus‐induced gene 2

The GPCR Epstein–Barr virus‐induced gene 2 (EBI2, also known as GPR183) is activated by oxysterols and plays a pivotal role in the regulation of B cell migration during immune responses. While the molecular basis of agonist binding has been addressed in several studies, the concept of biased agonism of the EBI2 receptor has not been explored.

Is It Time for New Thinking About High-Density Lipoprotein?

The perception of high-density lipoprotein (HDL) cholesterol as the good cholesterol that protects against atherosclerotic cardiovascular disease (ASCVD) has persisted for decades based largely on the countless observational studies showing high risk of ASCVD in individuals with low concentrations of HDL cholesterol.1 The notion has been the higher the better and the faith in HDL cholesterol–elevating drugs as a new treatment option for ASCVD so great, that >70 000 patients worldwide have been randomized in phase III outcome trials with the 4 major cholesteryl ester transfer protein inhibitors.2–5 Cholesteryl ester transfer protein inhibitors effectively increase HDL cholesterol, but to date none of these compounds have been marketed because of disappointing effects on the risk of ASCVD, and one drug even increased ASCVD risk as well as all-cause mortality.2 Despite the failures of HDL cholesterol–elevating drugs, which in addition to cholesteryl ester transfer protein inhibitors include niacin,6 and genetic studies showing that genetically elevated HDL cholesterol does not confer lower risk of ASCVD,7–12 the perception of HDL cholesterol as the good cholesterol is still deeply believed among many physicians, scientists, and individuals in the general public. See accompanying article on page 669 In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Hamer et al13 provide further data to …

Temporal trends in hip fracture incidence, mortality, and morbidity in Denmark from 1999 to 2012

Background and purpose — While development in hip fracture incidence and mortality is well examined, none has yet looked at the temporal trends regarding prevalence of co-morbidities. Therefore we investigated changes in incidence of first hip fracture, co-morbidity prevalence, 30 day- and 1-year mortality in hip fracture patients in the Danish population during the period 1999 to 2012. Patients and methods — Patients >18 years admitted with a fractured hip in Denmark between 1996 and 2012 were identified with data for the period 1999–2012 being analyzed regarding prevalence of co-morbidities, incidence, and mortality. Results — 122,923 patients were identified. Incidence in the whole population declined but sex-specific analysis showed no changes for men. For the whole study population, 30-day and 1-year mortality remained unchanged. Age at time of first hip fracture also remained unchanged. Of the included co-morbidities a decrease in prevalence of malignancy and dementia in women was found while there was an increase in the prevalence of all remaining co-morbidities, except hemi- or paraplegia for both sexes, rheumatic diseases for women, and for men diabetes with complications, myocardial infarction, AIDS/HIV, and malignancy. Interpretation — While hip fracture incidence declined for women it was unchanged for men; likewise, 30-day and 1-year mortality rates together with age at first fracture remained unchanged. When these results are compared with the relatively large increase in the prevalence of co-morbidities, it does not seem likely that the increased disease burden is affecting either the incidence or the mortality.

Hyperkalemia is Associated with Increased 30-Day Mortality in Hip Fracture Patients

Abnormal plasma concentrations of potassium in the form of hyper- and hypokalemia are frequent among hospitalized patients and have been linked to poor outcomes. In this study, we examined the prevalence of hypo- and hyperkalemia in patients admitted with a fractured hip as well as the association with 30-day mortality in these patients. A total of 7293 hip fracture patients (aged 60 years or above) with admission plasma potassium measurements were included. Data on comorbidity, medication, and death was retrieved from national registries. The association between plasma potassium and mortality was examined using Cox proportional hazards models adjusted for age, sex, and comorbidities. The prevalence of hypo- and hyperkalemia on admission was 19.8% and 6.6%, respectively. The 30-day mortality rates were increased for patients with hyperkalemia (21.0%, p < 0.0001) compared to normokalemic patients (9.5%), whereas hypokalemia was not significantly associated with mortality. After adjustment for age, sex, and individual comorbidities, hyperkalemia was still associated with increased risk of death 30 days after admission (HR = 1.93 [1.55–2.40], p < 0.0001). After the same adjustments, hypokalemia remained non-associated with increased risk of 30-day mortality (HR = 1.06 [0.87–1.29], p = 0.6). Hyperkalemia, but not hypokalemia, at admission is associated with increased 30-day mortality after a hip fracture.

Temporal trends in the use of antithrombotics at admission

Background and purpose — Currently, no clear evidence exists on the pattern of use of antithrombotics at admission in hip fracture patients and how this has changed over time. We investigated temporal trends in—and factors associated with—the use of antithrombotics in patients admitted with a fractured hip. Patients and methods — This was a population-based cohort study including all patients aged 18 years or above who were admitted with a hip fracture in Denmark from 1996 to 2012. The Danish national registries were used to collect information on medication use, vital status, and comorbidity. Results — From 1996 to 2012, the proportion of patients using antithrombotics in general increased by a factor of 2.3 from 19% to 43% (p < 0.001). More specifically, the use of anticoagulants increased by a factor of 6.8 and the use of antiplatelets increased by a factor of 2.1. When we adjusted for possible confounders, the use of antithrombotics still increased for every calendar year (relative risk (RR) = 1.03, CI: 1.03–1.04; p < 0.001). Age, sex, and Charlson comorbidity index were all associated with the use of antithrombotics (all p < 0.001). Interpretation — The proportion of hip fracture patients using antithrombotics at admission has increased substantially in Denmark over the last 2 decades. This highlights the need for evidence-based guidelines on how to handle patients using antithrombotics to ensure safe surgery and to avoid surgical delay.