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1.  Prenatal Valproate Exposure and Risk of Autism Spectrum Disorders and Childhood Autism 
JAMA  2013;309(16):1696-1703.
Importance
Valproate is used for the treatment of epilepsy and other neuropsychological disorders and may be the only treatment option for women of childbearing potential. However, prenatal exposure to valproate may increase the risk of autism.
Objective
To determine whether prenatal exposure to valproate is associated with an increased risk of autism in offspring.
Design, Setting, and Participants
Population-based study of all children born alive in Denmark from 1996 to 2006. National registers were used to identify children exposed to valproate during pregnancy and diagnosed with autism spectrum disorders (childhood autism [autistic disorder], Asperger syndrome, atypical autism, and other or unspecified pervasive developmental disorders). We analyzed the risks associated with all autism spectrum disorders as well as childhood autism. Data were analyzed by Cox regression adjusting for potential confounders (maternal age at conception, paternal age at conception, parental psychiatric history, gestational age, birth weight, sex, congenital malformations, and parity). Children were followed up from birth until the day of autism spectrum disorder diagnosis, death, emigration, or December 31, 2010, whichever came first.
Main Outcomes and Measures
Absolute risk (cumulative incidence) and the hazard ratio (HR) of autism spectrum disorder and childhood autism in children after exposure to valproate in pregnancy.
Results
Of 655 615 children born from 1996 through 2006, 5437 were identified with autism spectrum disorder, including 2067 with childhood autism. The mean age of the children at end of follow-up was 8.84 years (range, 4-14; median, 8.85). The estimated absolute risk after 14 years of follow-up was 1.53% (95% CI, 1.47%- 1.58%) for autism spectrum disorder and 0.48% (95% CI, 0.46%-0.51%) for childhood autism. Overall, the 508 children exposed to valproate had an absolute risk of 4.42% (95% CI, 2.59%-7.46%) for autism spectrum disorder (adjusted HR, 2.9 [95% CI, 1.7-4.9]) and an absolute risk of 2.50% (95% CI, 1.30%-4.81%) for childhood autism (adjusted HR, 5.2 [95% CI, 2.7-10.0]). When restricting the cohort to the 6584 children born to women with epilepsy, the absolute risk of autism spectrum disorder among 432 children exposed to valproate was 4.15% (95% CI, 2.20%-7.81%) (adjusted HR, 1.7 [95% CI, 0.9-3.2]), and the absolute risk of childhood autism was 2.95% (95% CI, 1.42%-6.11%) (adjusted HR, 2.9 [95% CI, 1.4-6.0]) vs 2.44% (95% CI, 1.88%-3.16%) for autism spectrum disorder and 1.02% (95% CI, 0.70%-1.49%) for childhood autism among 6152 children not exposed to valproate.
Conclusions and Relevance
Maternal use of valproate during pregnancy was associated with a significantly increased risk of autism spectrum disorder and childhood autism in the offspring, even after adjusting for maternal epilepsy. For women of childbearing potential who use antiepileptic medications, these findings must be balanced against the treatment benefits for women who require valproate for epilepsy control.
doi:10.1001/jama.2013.2270
PMCID: PMC4511955  PMID: 23613074
2.  Congenital cerebral palsy and prenatal exposure to self-reported maternal infections, fever, or smoking 
American journal of obstetrics and gynecology  2013;209(4):332.e1-332.e10.
OBJECTIVE
The objective of the study was to investigate the association between maternal self-reported infections, fever, and smoking in the prenatal period and the subsequent risk for congenital cerebral palsy (CP).
STUDY DESIGN
We included the 81,066 mothers of singletons born between 1996 and 2003 who participated in the Danish National Birth Cohort. Children were followed up through December 2008. Information on maternal infections, fever, smoking, and other demographic and lifestyle factors during pregnancy were reported by mothers in computer-assisted telephone interviews in early and midgestation. We identified 139 CP cases including 121 cases of spastic CP (sCP) as confirmed by the Danish National Cerebral Palsy Register. Cox proportional hazards regression models were used to estimate adjusted hazard ratios (aHRs) and 95% confidence intervals (CIs).
RESULTS
Self-reported vaginal infections were associated with an increased risk of CP and sCP (aHR, 1.52; 95% CI, 1.04–2.24; and aHR, 1.73; 95% CI, 1.16–2.60, respectively) and particularly untreated vaginal infections were associated with an increased risk of sCP (aHR, 1.95; 95% CI, 1.16–3.26). Fever was associated with the risk of CP (aHR, 1.53; 95% CI, 1.06–2.21). Smoking 10 or more cigarettes per day during pregnancy was also associated with sCP (aHR, 1.80; 95% CI, 1.10–2.94). There was a modest excess in risk for children exposed to both heavy smoking and vaginal infections. No other self-reported infections were significantly associated with CP.
CONCLUSION
Self-reported vaginal infections, fever, and smoking 10 or more cigarettes per day during pregnancy were associated with a higher risk of overall CP and/or sCP.
doi:10.1016/j.ajog.2013.06.023
PMCID: PMC4512233  PMID: 23791566
congenital cerebral palsy; maternal infections; pregnancy; smoking
3.  Risk of Fetal Death after Treatment with Antipsychotic Medications during Pregnancy 
PLoS ONE  2015;10(7):e0132280.
Background
Antipsychotic medications are increasingly used during pregnancy. Nevertheless, fetal risks are still not fully studied. It is currently unclear whether the antipsychotic treatment might induce a higher risk of fetal death. We aimed to determine if use of antipsychotic medication during pregnancy is associated with an increased risk of spontaneous abortion or stillbirth.
Methods
In a historical cohort study, we identified all clinically recognized pregnancies registered in the nationwide Danish registries from 1997 to 2008 (N = 1,005,319). Exposure was defined as any prescription of antipsychotic medications redeemed by the pregnant women during the exposure window, and recorded in the Danish National Prescription Register. Outcome was defined as any spontaneous abortion or stillbirth recorded in the Danish National Hospital Register and the Danish Medical Birth Register respectively.
Results
Women exposed to antipsychotic medications during pregnancy had a 34% higher risk of spontaneous abortion (adjusted relative risk = 1.34; 95% confidence interval = 1.22; 1.46) compared to unexposed women, but a similar risk compared to women exposed prior to (but not during) pregnancy (adjusted relative risk = 1.04; 95% confidence interval = 0.93; 1.17). The risk of spontaneous abortion was not increased in exposed pregnancies when compared to unexposed pregnancies in the same women (adjusted hazard ratio = 1.11; 95% CI = 0.94; 1.31). A twofold higher risk of stillbirth was found in women exposed to antipsychotic medications compared with unexposed women (relative risk = 2.27; 95% confidence interval = 1.45; 3.55) and compared with women exposed only prior to pregnancy (relative risk = 2.06; 95% confidence interval = 1.01; 4.19).
Conclusions
The increased risk of spontaneous abortion found in women treated with antipsychotic medications during pregnancy is most likely due to confounding factors. The risk of stillbirth was twofold higher in pregnancies exposed to antipsychotic medication during pregnancy. Treatment with antipsychotic medications during pregnancy requires careful consideration.
doi:10.1371/journal.pone.0132280
PMCID: PMC4498617  PMID: 26162087
4.  Characterization of an anti-Bla g 1 scFv: Epitope mapping and cross-reactivity 
Molecular immunology  2014;59(2):200-207.
Bla g 1 is a major allergen from Blatella germanica and one of the primary allergens used to assess cockroach allergen exposure. The epitope of an anti-Bla g 1 scFv was mapped in order to better understand cross reactivity with other group 1 cockroach allergens and patient IgE epitopes. X-ray crystallography was used to determine the structure of the scFv. The scFv epitope on Bla g 1 was located by alanine scanning site-directed mutagenesis and ELISA. Twenty-six rBla g 1-GST alanine mutants were evaluated for variations in binding to the scFv compared to the wild type allergen. Six mutants showed a significant difference in scFv binding affinity. These mutations clustered to form a discontinuous epitope mainly comprising two helices of Bla g 1. The allergen-scFv complex was modeled based on the results, and the epitope region was found to have low sequence similarity with Per a 1, especially among the residues identified as functionally important for the scFv binding to Bla g 1. Indeed, the scFv failed to bind Per a 1 in American cockroach extract. The scFv was unable to inhibit the binding of IgE antibodies from a highly cockroach allergic patient to Bla g 1. Based on the surface area of Bla g 1 occluded by the scFv, putative regions of patient IgE–Bla g 1 interactions can be inferred. This scFv could be best utilized as a capture antibody in an IgE detection ELISA, or to differentiate Bla g 1 from Per a 1 in environmental exposure assays.
doi:10.1016/j.molimm.2014.02.003
PMCID: PMC4097036  PMID: 24667070
Allergen; Structure; Bla g 1; scFv; Cockroach; Epitope
5.  Disproportionate Fetal Growth and the Risk for Congenital Cerebral Palsy in Singleton Births 
PLoS ONE  2015;10(5):e0126743.
Objective
To investigate the association between proportionality of fetal and placental growth measured at birth and the risk for congenital cerebral palsy (CP).
Study Design
We identified all live-born singletons born in Denmark between 1995 and 2003 and followed them from 1 year of age until December 31st, 2008. Information on four indices of fetal growth: ponderal index, head circumference/ abdominal circumference ratio, cephalization index and birth weight/ placenta weight ratio was collected. Cox proportional hazards regression models were used to estimate adjusted hazard ratios (aHR) and 95% confidence intervals (CI). All measurements were evaluated as gestational age and sex specific z-scores and in z-score percentile groups, adjusted for potential confounders, and stratified on gestational age groups (<32, 32-36, 37-38, 39, 40, ≥41 weeks).
Results
We identified 503,784 singleton births, of which 983 were confirmed cases of CP. Head/ abdominal circumference ratio (aHR:1.12; 95%CI:1.07-1.16) and cephalization index (aHR:1.14; 95%CI:1.11-1.16) were associated with the risk of CP irrespective of gestational age. Birth weight-placental weight ratio was also associated with CP in the entire cohort (aHR:0.90; 95%CI:0.83-0.97). Ponderal index had a u-shaped association with CP, where both children with low and high ponderal index were at higher risk of CP.
Conclusions
CP is associated with disproportions between birth weight, birth length, placental weight and head circumference suggesting pre and perinatal conditions contribute to fetal growth restriction in children with CP.
doi:10.1371/journal.pone.0126743
PMCID: PMC4431832  PMID: 25974407
6.  The Danish National Prescription Registry in studies of a biological pharmaceutical: palivizumab – validation against two external data sources 
Clinical Epidemiology  2015;7:305-312.
Background
National prescription databases are important tools in pharmacoepidemiological studies investigating potential long-term adverse events after drug use. Palivizumab is a biological pharmaceutical used as passive prophylaxis against severe infection with respiratory syncytial virus in high-risk children.
Objective
To assess the registration of palivizumab in the Danish National Prescription Registry (DNPR) and to examine if palivizumab reimbursement data obtained from the Danish Health and Medicines Authority could serve as a supplement to data from the DNPR.
Methods
Registration of palivizumab exposure in the DNPR between 1999 and 2010 was compared to two external data sources: registration of palivizumab exposure in medical records, and palivizumab reimbursement data.
Results
During the study period, 182 children with palivizumab exposure were registered in the DNPR. A total of 207 children were registered for palivizumab reimbursement. The sensitivity of palivizumab registration in the DNPR was 26% (20%–34%), and the specificity of no palivizumab registration in the DNPR was 97% (94%–99%), with data from the medical record as the reference. Palivizumab registration sensitivity in reimbursement data was 29% (22%–36%), and the specificity of no palivizumab registration in the DNPR was 97% (94%–99%), with data from the medical record as the reference.
Conclusion
Exposure to palivizumab was underestimated in the DNPR. Reimbursement data are a readily accessible data supplement, which only slightly increased the sensitivity of palivizumab registration in the DNPR. Our findings underline the need to improve DNPR information concerning drugs administered in hospitals.
doi:10.2147/CLEP.S73355
PMCID: PMC4431470  PMID: 26056490
drug; health register; medical records; respiratory syncytial virus; validation
7.  Structure–Function Studies of DNA Polymerase λ 
Biochemistry  2014;53(17):2781-2792.
DNA polymerase λ (pol λ) functions in DNA repair with its main roles considered to be filling short gaps during repair of double-strand breaks by nonhomologous end joining and during base excision repair. As indicated by structural and biochemical studies over the past 10 years, pol λ shares many common properties with other family X siblings (pol β, pol μ, and terminal deoxynucleotidyl transferase) but also has unique structural features that determine its specific functions. In this review, we consider how structural studies over the past decade furthered our understanding of the behavior and biological roles of pol λ.
doi:10.1021/bi4017236
PMCID: PMC4018081  PMID: 24716527
8.  Impact of cardiovascular risk factors and medication use on the efficacy of remote ischaemic conditioning: post hoc subgroup analysis of a randomised controlled trial 
BMJ Open  2015;5(4):e006923.
Objectives
Remote ischaemic conditioning (RIC) promotes cardioprotection in patients undergoing primary percutaneous coronary intervention (pPCI) for ST-elevation myocardial infarction (STEMI). The effect of RIC may be modified by cardiovascular risk factors and their medications. We examined whether cardiovascular risk factors, lipid and glucose levels, and medication use influenced the efficacy of RIC in patients with STEMI treated with pPCI.
Design
Post hoc subgroup analysis of a single-centre randomised controlled trial.
Participants
A total of 139 patients with STEMI, randomised during ambulance transport to hospital for pPCI with (n=71) or without (n=68) RIC, met the trial criteria and achieved data for a myocardial salvage index (MSI).
Interventions
RIC was administered through intermittent arm ischaemia with four cycles of 5 min inflation and 5 min deflation of a blood pressure cuff.
Primary outcome measures
MSI, estimated by single-photon emission CT. We evaluated the efficacy of RIC on the MSI in patient subgroups of cardiovascular risk factors, lipid and glucose levels, and medication use.
Results
We found no significant difference in the efficacy of RIC in subgroups of cardiovascular risk factors, lipid and glucose levels, and medication use. However, point estimates indicated a reduced effect of RIC among smokers (median difference in MSI between RIC and control groups: −0.02 (95% CI −0.32 to 0.28) in smokers vs 0.25 (95% CI 0.08 to 0.42) in non-smokers, p value for interaction=0.13) and an increased effect of RIC in statin users (median difference in MSI between RIC and control groups: 0.34 (95% CI 0.03 to 0.65) in statin users vs 0.09 (95% CI −0.11 to 0.29) in non-statin users, p value for interaction=0.19).
Conclusions
RIC as an adjunct to pPCI seems to improve MSI in our trial population of patients with STEMI regardless of most cardiovascular risk factors and their medications. Our post hoc finding on a limited sample size calls for further investigation in large-scale multicentre trials.
Trial registration number
NCT00435266.
doi:10.1136/bmjopen-2014-006923
PMCID: PMC4390720  PMID: 25838505
9.  Immunostimulant patches containing Escherichia coli LT enhance immune responses to DNA- and recombinant protein-based Alzheimer’s disease vaccines 
Journal of neuroimmunology  2014;268(0):50-57.
Immunotherapeutic approaches to treating Alzheimer’s disease (AD) using vaccination strategies must overcome the obstacle of achieving adequate responses to vaccination in the elderly. Here we demonstrate for the first time that application of the E. coli heat-labile enterotoxin adjuvant-laden immunostimulatory patches (LT-IS) dramatically enhances the onset and magnitude of immune responses to DNA- and protein-based vaccines for Alzheimer’s disease following intradermal immunization via gene gun and conventional needles, respectively. Our studies suggest that the immune activation mediated by LT-IS offers improved potency for generating AD-specific vaccination responses that should be investigated as an adjuvant in the clinical arena.
doi:10.1016/j.jneuroim.2014.01.002
PMCID: PMC3951952  PMID: 24507620
Alzheimer’s disease; epitope vaccine; immunostimulatory patch; immune responses
10.  Impact of preadmission treatment with calcium channel blockers or beta blockers on short-term mortality after stroke: a nationwide cohort study 
BMC Neurology  2015;15:24.
Background
The prognostic impact of preadmission use of calcium channel blockers (CCBs) and beta blockers (BBs) on stroke mortality remains unclear. We aimed to examine whether preadmission use of CCBs or BBs was associated with improved short-term mortality following ischemic stroke, intracerebral hemorrhage (ICH), or subarachnoid hemorrhage (SAH).
Methods
We conducted a nationwide population-based cohort study using Danish medical registries. We identified all patients with a first-time inpatient diagnosis of stroke between 2004 and 2012 and their comorbidities. We defined CCB/BB use as current use, former use, or non-use. Current use was further classified as new or long-term use. We used Cox regression modeling to compute 30-day mortality rate ratios (MRRs) with 95% confidence intervals (CIs), controlling for potential confounders.
Results
We identified 100,043 patients with a first-time stroke. Of these, 83,736 (83.7%) patients had ischemic stroke, 11,779 (11.8%) had ICH, and 4,528 (4.5%) had SAH. Comparing current users of CCBs or BBs with non-users, we found no association with mortality for ischemic stroke [adjusted 30-day MRR = 0.99 (95% CI: 0.94-1.05) for CCBs and 1.01 (95% CI: 0.96-1.07) for BBs], ICH [adjusted 30-day MRR = 1.05 (95% CI: 0.95-1.16) for CCBs and 0.95 (95% CI: 0.87-1.04) for BBs], or SAH [adjusted 30-day MRR = 1.05 (95% CI: 0.85-1.29) for CCBs and 0.89 (95% CI: 0.72-1.11) for BBs]. Former use of CCBs or BBs was not associated with mortality.
Conclusions
Preadmission use of CCBs or BBs was not associated with 30-day mortality following ischemic stroke, ICH, or SAH.
Electronic supplementary material
The online version of this article (doi:10.1186/s12883-015-0279-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12883-015-0279-3
PMCID: PMC4365558  PMID: 25884780
Beta-blocker; Calcium channel blocker; Stroke; Ischemic stroke; Hemorrhagic stroke; Mortality
11.  Sustained active site rigidity during synthesis by human DNA polymerase μ 
DNA polymerase mu (Pol μ) is the only template-dependent human DNA polymerase capable of repairing double strand DNA breaks (DSBs) with unpaired 3′-ends in non-homologous end joining (NHEJ). To probe this function, we structurally characterized Pol μ’s catalytic cycle for single nucleotide incorporation. These structures indicate that, unlike other template-dependent DNA polymerases, there are no large-scale conformational changes in protein subdomains, amino acid side chains, or DNA upon dNTP binding or catalysis. Instead, the only major conformational change is seen earlier in the catalytic cycle, when the flexible Loop1 region repositions upon DNA binding. Pol μ variants with changes in Loop1 have altered catalytic properties and are partially defective in NHEJ. The results indicate that specific Loop1 residues contribute to Pol μ’s unique ability to catalyze template-dependent NHEJ of DSBs with unpaired 3′-ends.
doi:10.1038/nsmb.2766
PMCID: PMC4164209  PMID: 24487959
Family X DNA polymerase; nonhomologous end joining; DNA double strand break repair; Loop1
12.  Diversity Outbred Mice Identify Population-Based Exposure Thresholds and Genetic Factors that Influence Benzene-Induced Genotoxicity 
Environmental Health Perspectives  2014;123(3):237-245.
Background
Inhalation of benzene at levels below the current exposure limit values leads to hematotoxicity in occupationally exposed workers.
Objective
We sought to evaluate Diversity Outbred (DO) mice as a tool for exposure threshold assessment and to identify genetic factors that influence benzene-induced genotoxicity.
Methods
We exposed male DO mice to benzene (0, 1, 10, or 100 ppm; 75 mice/exposure group) via inhalation for 28 days (6 hr/day for 5 days/week). The study was repeated using two independent cohorts of 300 animals each. We measured micronuclei frequency in reticulocytes from peripheral blood and bone marrow and applied benchmark concentration modeling to estimate exposure thresholds. We genotyped the mice and performed linkage analysis.
Results
We observed a dose-dependent increase in benzene-induced chromosomal damage and estimated a benchmark concentration limit of 0.205 ppm benzene using DO mice. This estimate is an order of magnitude below the value estimated using B6C3F1 mice. We identified a locus on Chr 10 (31.87 Mb) that contained a pair of overexpressed sulfotransferases that were inversely correlated with genotoxicity.
Conclusions
The genetically diverse DO mice provided a reproducible response to benzene exposure. The DO mice display interindividual variation in toxicity response and, as such, may more accurately reflect the range of response that is observed in human populations. Studies using DO mice can localize genetic associations with high precision. The identification of sulfotransferases as candidate genes suggests that DO mice may provide additional insight into benzene-induced genotoxicity.
Citation
French JE, Gatti DM, Morgan DL, Kissling GE, Shockley KR, Knudsen GA, Shepard KG, Price HC, King D, Witt KL, Pedersen LC, Munger SC, Svenson KL, Churchill GA. 2015. Diversity Outbred mice identify population-based exposure thresholds and genetic factors that influence benzene-induced genotoxicity. Environ Health Perspect 123:237–245; http://dx.doi.org/10.1289/ehp.1408202
doi:10.1289/ehp.1408202
PMCID: PMC4348743  PMID: 25376053
13.  Adverse pregnancy outcomes after exposure to methylphenidate or atomoxetine during pregnancy 
Clinical Epidemiology  2015;7:139-147.
Objective
To determine if prenatal exposure to methylphenidate (MPH) or atomoxetine (ATX) increases the risk of adverse pregnancy outcomes in women with attention deficit/hyperactivity disorder (ADHD).
Materials and methods
This was a population-based cohort study of all pregnancies in Denmark from 1997 to 2008. Information on use of ADHD medication, ADHD diagnosis, and pregnancy outcomes was obtained from nationwide registers.
Results
We identified 989,932 pregnancies, in which 186 (0.02%) women used MPH/ATX and 275 (0.03%) women had been diagnosed with ADHD but who did not take MPH/ATX. Our reference pregnancies had no exposure to MPH/ATX and no ADHD diagnosis. Exposure to MPH/ATX was associated with an increased risk of spontaneous abortion (SA; ie, death of an embryo or fetus in the first 22 weeks of gestation) (adjusted relative risk [aRR] 1.55, 95% confidence interval [CI] 1.03–2.36). The risk of SA was also increased in pregnancies where the mother had ADHD but did not use MPH/ATX (aRR 1.56, 95% CI 1.11–2.20). The aRR of Apgar scores <10 was increased among exposed women (aRR 2.06, 95% CI 1.11–3.82) but not among unexposed women with ADHD (aRR 0.99, 95% CI 0.48–2.05).
Conclusion
MPH/ATX was associated with a higher risk of SA, but our study indicated that it may at least partly be explained by confounding by indication. Treatment with MPH/ATX was however associated with low Apgar scores <10, an association not found among women with ADHD who did not use MPH/ATX.
doi:10.2147/CLEP.S72906
PMCID: PMC4317061  PMID: 25657597
attention deficit/hyperactivity disorder; ADHD; methylphenidate; atomoxetine; pregnancy outcomes
14.  The Western Denmark Cardiac Computed Tomography Registry: a review and validation study 
Clinical Epidemiology  2014;7:53-64.
Background
As a subregistry to the Western Denmark Heart Registry (WDHR), the Western Denmark Cardiac Computed Tomography Registry (WDHR-CCTR) is a clinical database established in 2008 to monitor and improve the quality of cardiac computed tomography (CT) in Western Denmark.
Objective
We examined the content, data quality, and research potential of the WDHR-CCTR.
Methods
We retrieved 2008–2012 data to examine the 1) content; 2) completeness of procedure registration using the Danish National Patient Registry as reference; 3) completeness of variable registration comparing observed vs expected numbers; and 4) positive predictive values as well as negative predictive values of 19 main patient and procedure variables.
Results
By December 31, 2012, almost 22,000 cardiac CTs with up to 40 variables for each procedure have been registered. Of these, 87% were coronary CT angiography performed in patients with symptoms indicative of coronary artery disease. Compared with the Danish National Patient Registry, the overall procedure completeness was 72%. However, an additional medical record review of 282 patients registered in the Danish National Patient Registry, but not in the WDHR-CCTR, showed that coronary CT angiographies accounted for only 23% of all nonregistered cardiac CTs, indicating >90% completeness of coronary CT angiographies in the WDHR-CCTR. The completeness of individual variables varied substantially (range: 0%–100%), but was >85% for more than 70% of all variables. Using medical record review of 250 randomly selected patients as reference standard, the positive predictive value for the 19 variables ranged from 89% to 100% (overall 97%), whereas the negative predictive value ranged from 97% to 100% (overall 99%). Stratification by center status showed consistently high positive and negative predictive values for both university (96%/99%) and nonuniversity centers (97%/99%).
Conclusion
WDHR-CCTR provides ongoing prospective registration of all cardiac CTs performed in Western Denmark since 2008. Overall, the registry data have a high degree of completeness and validity, making it a valuable tool for clinical epidemiological research.
doi:10.2147/CLEP.S73728
PMCID: PMC4317160  PMID: 25657592
coronary computed tomography angiography; database; epidemiology; registries
15.  Novel structure of cockroach allergen Bla g 1 has implications for allergenicity and exposure assessment 
The Journal of allergy and clinical immunology  2013;132(6):10.1016/j.jaci.2013.06.014.
Background
Sensitization to cockroach allergens is a major risk factor for asthma. The cockroach allergen Bla g 1 has multiple repeats of ~100 amino acids, but the fold of the protein and the biological function are unknown.
Objective
To determine the structure of Bla g 1, investigate the implications for allergic disease, and standardize cockroach exposure assays.
Methods
Natural Bla g 1 and recombinant constructs were compared by ELISA using specific murine IgG and human IgE. The structure of Bla g 1 was determined by X-ray crystallography. Mass spectrometry and NMR were utilized to examine ligand-binding properties of the allergen.
Results
The structure of a recombinant Bla g 1 construct with comparable IgE and IgG reactivity to the natural allergen was solved by X-ray crystallography. The Bla g 1 repeat forms a novel fold with 6 helices. Two repeats encapsulate a large and nearly spherical hydrophobic cavity, defining the basic structural unit. Lipids in the cavity varied depending on the allergen origin. Palmitic, oleic and stearic acids were associated with nBla g 1 from cockroach frass. One Unit of Bla g 1 was equivalent to 104 ng of allergen.
Conclusions
Bla g 1 has a novel fold with a capacity to bind various lipids, which suggests a digestive function associated with non-specific transport of lipid molecules in cockroaches. Defining the basic structural unit of Bla g 1 facilitates the standardization of assays in absolute units for the assessment of environmental allergen exposure.
doi:10.1016/j.jaci.2013.06.014
PMCID: PMC3844097  PMID: 23915714
Allergen; asthma; Bla g 1; cockroach; structure; ligand-binding proteins; exposure assessment
16.  Structural characterization of the virulence factor nuclease A from Streptococcus agalactiae  
Nuclease A (NucA) is an extracellular nuclease secreted from S. agalactiae and is required for full virulence during infection. Crystal structures and biochemical characterization of NucA mutants reveal possible roles for surface residues in DNA substrate binding and catalysis. These results may serve as a foundation for the design of targeted antibacterial therapeutic compounds.
The group B pathogen Streptococcus agalactiae commonly populates the human gut and urogenital tract, and is a major cause of infection-based mortality in neonatal infants and in elderly or immunocompromised adults. Nuclease A (GBS_NucA), a secreted DNA/RNA nuclease, serves as a virulence factor for S. agalactiae, facilitating bacterial evasion of the human innate immune response. GBS_NucA efficiently degrades the DNA matrix component of neutrophil extracellular traps (NETs), which attempt to kill and clear invading bacteria during the early stages of infection. In order to better understand the mechanisms of DNA substrate binding and catalysis of GBS_NucA, the high-resolution structure of a catalytically inactive mutant (H148G) was solved by X-ray crystallography. Several mutants on the surface of GBS_NucA which might influence DNA substrate binding and catalysis were generated and evaluated using an imidazole chemical rescue technique. While several of these mutants severely inhibited nuclease activity, two mutants (K146R and Q183A) exhibited significantly increased activity. These structural and biochemical studies have greatly increased our understanding of the mechanism of action of GBS_NucA in bacterial virulence and may serve as a foundation for the structure-based drug design of antibacterial compounds targeted to S. agalactiae.
doi:10.1107/S1399004714019725
PMCID: PMC4220975  PMID: 25372684
nuclease A; virulence factors; Streptococcus agalactiae
17.  Acromegaly according to the Danish National Registry of Patients: how valid are ICD diagnoses and how do patterns of registration affect the accuracy of registry data? 
Clinical Epidemiology  2014;6:295-299.
Background
The incidence of acromegaly is uncertain, since population-based studies are few. In the absence of a specific acromegaly registry, the Danish National Registry of Patients (DNRP) becomes a potential source of data for studying the epidemiology of acromegaly, by linking all hospital discharge diagnoses to the personal identification numbers of individual Danish inhabitants. The validity of the DNRP with respect to acromegaly, however, remains to be tested. The aim of this study was to validate the International Classification of Diseases (ICD) codes for acromegaly (ICD-8: 25300, 25301. ICD-10: E22.0) as used in the DNRP, and to assess the influence of various registration patterns on the accuracy of registry data.
Methods
We identified patients registered with ICD codes for the diagnosis of acromegaly or other pituitary disorders during the period 1991–2009. Data on the institutional origin of each registration and the number of relevant DNRP registrations were recorded, and systematic patient chart reviews were performed to confirm the diagnosis.
Results
In total, 110 cases of acromegaly were confirmed, compared with 275 registered cases, yielding a positive predictive value (PPV) of 40%. When restricting the search to the regional highly specialized department of endocrinology, the PPV increased to 53% with no loss of cases with confirmed acromegaly. With a requirement of at least one, two, or three DNRP registrations, the PPV increased, but with a concurrent loss of confirmed cases.
Conclusion
The DNRP seems to be a useful source for identifying new cases of acromegaly, especially when restricting the search to a relevant regional highly specialized department. The PPV of DNRP data used for this purpose can be increased by including only cases with several registrations. A similar approach may be successfully applied to other rare diseases in which continuity of care is provided by highly specialized departments.
doi:10.2147/CLEP.S63758
PMCID: PMC4155997  PMID: 25210475
acromegaly; Danish National Registry of Patients; validation; epidemiology; incidence
18.  Enzymatic Redesigning of Biologically Active Heparan Sulfate* 
The Journal of biological chemistry  2005;280(52):42817-42825.
Heparan sulfate carries a wide range of biological activities, regulating blood coagulation, cell differentiation, and inflammatory responses. The sulfation patterns of the polysaccharide are essential for the biological activities. In this study, we report an enzymatic method for the sulfation of multimilligram amounts of heparan sulfate with specific functions using immobilized sulfotransferases combined with a 3′-phosphoadenosine 5′-phosphosulfate regeneration system. By selecting appropriate enzymatic modification steps, an inactive precursor has been converted to the heparan sulfate having three distinct biological activities, associated with binding to antithrombin, fibroblast growth factor-2, and herpes simplex virus envelope glycoprotein D. Because the recombinant sulfotransferases are expressed in bacteria, and the method uses a low cost sulfo donor, it can be readily utilized to synthesize large quantities of anticoagulant heparin drug or other biologically active heparan sulfates.
doi:10.1074/jbc.M504338200
PMCID: PMC4140617  PMID: 16260789
19.  Atrial Fibrillation as a Marker of Occult Cancer 
PLoS ONE  2014;9(8):e102861.
Background
Recent studies suggest that cancer increases risk of atrial fibrillation. Whether atrial fibrillation is a marker for underlying occult cancer is unknown.
Methods
We conducted a cohort study (1980–2011) of all Danish patients with new-onset atrial fibrillation. To examine cancer risk, we computed absolute risk at 3 months and standardized incidence ratios (SIRs) by comparing observed cancer incidence among patients newly diagnosed with atrial fibrillation with that expected based on national cancer incidence during the period.
Results
Median follow-up time was 3.4 years among 269 742 atrial fibrillation patients. Within 3 months of follow-up, 6656 cancers occurred (absolute risk, 2.5%; 95% confidence intervals [CI], 2.4%–2.5%) versus 1302 expected, yielding a SIR of 5.11; 95% CI, 4.99–5.24. Associations were particularly strong for cancers of the lung, kidney, colon, ovary, and for non-Hodgkin's lymphoma. The SIR within 3 months of follow-up was 7.02; 95% CI, 6.76–7.28 for metastatic and 3.53; 95% CI, 3.38–3.68 for localized cancer. Beyond 3 months of follow-up, overall cancer risk was modestly increased (SIR, 1.13; 95% CI, 1.12–1.15).
Conclusion
Patients with new-onset atrial fibrillation had a markedly increased relative risk of a cancer diagnosis within the next three months, however, corresponding absolute risk was small.
doi:10.1371/journal.pone.0102861
PMCID: PMC4138009  PMID: 25119880
20.  Structural Analysis of the Sulfotransferase (3-O-Sulfotransferase Isoform 3) Involved in the Biosynthesis of an Entry Receptor for Herpes Simplex Virus 1* 
The Journal of biological chemistry  2004;279(43):45185-45193.
Heparan sulfate (HS) plays essential roles in assisting herpes simplex virus infection and other biological processes. The biosynthesis of HS includes numerous specialized sulfotransferases that generate a variety of sulfated saccharide sequences, conferring the selectivity of biological functions of HS. We report a structural study of human HS 3-O-sulfotransferase isoform 3 (3-OST-3), a key sulfotransferase that transfers a sulfuryl group to a specific glucosamine in HS generating an entry receptor for herpes simplex virus 1. We have obtained the crystal structure of 3-OST-3 at 1.95 Å in a ternary complex with 3′-phosphoadenosine 5′-phosphate and a tetrasaccharide substrate. Mutational analyses were also performed on the residues involved in the binding of the substrate. Residues Gln255 and Lys368 are essential for the sulfotransferase activity and lie within hydrogen bonding distances to the carboxyl and sulfo groups of the uronic acid unit. These residues participate in the substrate recognition of 3-OST-3. This structure provides atomic level evidence for delineating the substrate recognition and catalytic mechanism for 3-OST-3.
doi:10.1074/jbc.M405013200
PMCID: PMC4114238  PMID: 15304505
21.  Stable RAGE-Heparan Sulfate Complexes are Essential for Signal Transduction 
ACS chemical biology  2013;8(7):1611-1620.
RAGE (Receptor for Advanced Glycation End-Products) has emerged as a major receptor that mediates vascular inflammation. Signaling through RAGE by damage-associated molecular pattern molecules often leads to uncontrolled inflammation that exacerbates the impact of the underlying disease. Oligomerization of RAGE is believed to play an essential role in signal transduction, but the molecular mechanism of oligomerization remains elusive. Here we report that RAGE activation of Erk1/2 phosphorylation on endothelial cells in response to a number of ligands depends on a mechanism that involves heparan sulfate-induced hexamerization of the RAGE extracellular domain. Structural studies of the extracellular V-C1 domain-dodecasaccharide complex by X-ray diffraction and small-angle X-ray scattering revealed that the hexamer consists of a trimer of dimers, with a stoichiometry of 2:1 RAGE:dodecasaccharide. Mutagenesis studies mapped the heparan sulfate binding site and the interfacial surface between the monomers, and demonstrated that electrostatic interactions with heparan sulfate and inter-monomer hydrophobic interactions work in concert to stabilize the dimer. The importance of oligomerization was demonstrated by inhibition of signaling with a new epitope-defined monoclonal antibody that specifically targets oligomerization. These findings indicate that RAGE-heparan sulfate oligomeric complexes are essential for signaling and that interfering with RAGE oligomerization might be of therapeutic value.
doi:10.1021/cb4001553
PMCID: PMC3806902  PMID: 23679870
22.  Use of β-Blockers, Angiotensin-Converting Enzyme Inhibitors, Angiotensin II Receptor Blockers, and Risk of Breast Cancer Recurrence: A Danish Nationwide Prospective Cohort Study 
Journal of Clinical Oncology  2013;31(18):2265-2272.
Purpose
To estimate associations between use of β-blockers, angiotensin-converting enzyme (ACE) inhibitors, or angiotensin receptor blockers (ARBs) and breast cancer recurrence in a large Danish cohort.
Patients and Methods
We enrolled 18,733 women diagnosed with nonmetastatic breast cancer between 1996 and 2003. Patient, treatment, and 10-year recurrence data were ascertained from the Danish Breast Cancer Cooperative Group registry. Prescription and medical histories were ascertained by linkage to the National Prescription Registry and Registry of Patients, respectively. β-Blocker exposure was defined in aggregate and according to solubility, receptor selectivity, and individual drugs. ACE inhibitor and ARB exposures were defined in aggregate. Recurrence associations were estimated with multivariable Cox regression models in which time-varying drug exposures were lagged by 1 year.
Results
Compared with never users, users of any β-blocker had a lower recurrence hazard in unadjusted models (unadjusted hazard ratio [HR] = 0.91; 95% CI, 0.81 to 1.0) and a slightly higher recurrence hazard in adjusted models (adjusted HR = 1.3; 95% CI, 1.1 to 1.5). Associations were similar for exposures defined by receptor selectivity and solubility. Although most individual β-blockers showed no association with recurrence, metoprolol and sotalol were associated with increased recurrence rates (adjusted metoprolol HR = 1.5, 95% CI, 1.2 to 1.8; adjusted sotalol HR = 2.0, 95% CI, 0.99 to 4.0). ACE inhibitors were associated with a slightly increased recurrence hazard, whereas ARBs were not associated with recurrence (adjusted ACE inhibitor HR = 1.2, 95% CI, 0.97 to 1.4; adjusted ARBs HR = 1.1, 95% CI, 0.85 to 1.3).
Conclusion
Our data do not support the hypothesis that β-blockers attenuate breast cancer recurrence risk.
doi:10.1200/JCO.2012.43.9190
PMCID: PMC3677839  PMID: 23650417
23.  Amino acid substitution in the active site of DNA polymerase β explains the energy barrier of the nucleotidyl transfer reaction 
DNA polymerase β (pol β) is a bifunctional enzyme widely studied for its roles in base excision DNA repair where one key function is gap-filling DNA synthesis. In spite of significant progress in recent years, the atomic level mechanism of the DNA synthesis reaction has remained poorly understood. Based on crystal structures of pol β in complex with its substrates and theoretical considerations of amino acids and metals in the active site, we have proposed that a nearby carboxylate group of Asp256 enables the reaction by accepting a proton from the primer O3′ group, thus activating O3′ as the nucleophile in the reaction path. Here, we tested this proposal by altering the side chain of Asp256 to Glu and then exploring the impact of this conservative change on the reaction. The D256E enzyme is more than 1,000-fold less active than the wild-type enzyme, and the crystal structures are subtly different in the active sites of the D256E and wild-type enzymes. Theoretical analysis of DNA synthesis by the D256E enzyme shows that the O3′ proton still transfers to the nearby carboxylate of residue 256. However, the electrostatic stabilization and location of the O3′ proton transfer during the reaction path are dramatically altered compared with wild-type. Surprisingly, this is due to repositioning of the Arg254 side chain in the Glu256 enzyme active site, such that Arg254 is not in position to stabilize the proton transfer from O3′. The theoretical results with the wild-type enzyme indicate early charge reorganization associated with the O3′ proton transfer, and this does not occur in the D256E enzyme. The charge reorganization is mediated by the catalytic magnesium ion in the active site.
doi:10.1021/ja403842j
PMCID: PMC3918438  PMID: 23647366
25.  Existing data sources for clinical epidemiology: Aarhus University Clinical Trial Candidate Database, Denmark 
Clinical Epidemiology  2014;6:129-135.
Denmark is facing a reduction in clinical trial activity as the pharmaceutical industry has moved trials to low-cost emerging economies. Competitiveness in industry-sponsored clinical research depends on speed, quality, and cost. Because Denmark is widely recognized as a region that generates high quality data, an enhanced ability to attract future trials could be achieved if speed can be improved by taking advantage of the comprehensive national and regional registries. A “single point-of-entry” system has been established to support collaboration between hospitals and industry. When assisting industry in early-stage feasibility assessments, potential trial participants are identified by use of registries to shorten the clinical trial startup times. The Aarhus University Clinical Trial Candidate Database consists of encrypted data from the Danish National Registry of Patients allowing an immediate estimation of the number of patients with a specific discharge diagnosis in each hospital department or outpatient specialist clinic in the Central Denmark Region. The free access to health care, thorough monitoring of patients who are in contact with the health service, completeness of registration at the hospital level, and ability to link all databases are competitive advantages in an increasingly complex clinical trial environment.
doi:10.2147/CLEP.S60080
PMCID: PMC3986109  PMID: 24748818
Denmark; single point-of-entry; patient registration

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