Background: Brominated flame retardants (BFRs), used in many types of consumer goods, are being studied because of concerns about possible health effects related to endocrine disruption, immunotoxicity, reproductive toxicity, and neurotoxicity. Tetrabromobisphenol A (TBBPA), the most widely used BFR, and human metabolites of certain congeners of polybrominated diphenyl ether (e.g., 3-OH-BDE-47) have been suggested to inhibit estrogen sulfotransferase, potentially affecting estrogen metabolism.
Objectives: Our primary goal was to understand the structural mechanism for inhibition of the hormone-metabolizing enzyme estrogen sulfotransferase by certain BFRs. We also sought to understand various factors that facilitate the binding of flame retardants in the enzyme binding pocket.
Methods: We used X-ray crystallography to obtain atomic detail of the binding modes of TBBPA and 3-OH-BDE-47 to estrogen sulfotransferase for comparison with binding of the endogenous substrate estradiol.
Results: The crystal structures reveal how BFRs mimic estradiol binding as well as the various interactions between the compounds and protein residues that facilitate its binding. In addition, the structures provide insights into the ability of the sulfotransferase substrate binding pocket to accommodate a range of halogenated compounds that satisfy minimal structural criteria.
Conclusions: Our results show how BFRs or their metabolites can bind to and inhibit a key hormone-metabolizing enzyme, potentially causing endocrine disruption.
Citation: Gosavi RA, Knudsen GA, Birnbaum LS, Pedersen LC. 2013. Mimicking of estradiol binding by flame retardants and their metabolites: a crystallographic analysis. Environ Health Perspect 121:1194–1199; http://dx.doi.org/10.1289/ehp.1306902
Alzheimer’s disease (AD) process is understood to involve the accumulation of amyloid plaques and tau tangles in the brain. However, attempts at targeting the main culprits, neurotoxic Aβ peptides, have thus far proven unsuccessful for improving cognitive function. Recent clinical trials with passively administrated anti-Aβ antibodies failed to slow cognitive decline in mild-moderate AD patients, but suggest that an immunotherapeutic approach could be effective in patients with mild AD. In an AD mouse model (Tg2576) we tested the immunogenicity (cellular and humoral immune responses) and efficacy (AD-like pathology) of clinical grade Lu AF20513 vaccine. Lu AF20513 induces robust “non-self” T cell responses and production of anti-Aβ antibodies that reduce AD-like pathology in the brains of Tg2576 mice without inducing microglial activation and enhancing astrocytosis or CAA. Importantly, a single immunization with Lu AF20513 induces strong humoral immunity in mice with pre-existing memory Th cells. In addition, Lu AF20513 induces strong humoral responses in guinea pigs and monkeys. Collectively, these data suggest translation of Lu AF20513 to clinical setting with aims to (i) induce therapeutically potent anti-Aβ antibody responses in patients with mild AD, particularly if they have memory Th cells generated after immunizations with conventional Tetanus Toxoid vaccine; (ii) exclude likely pathological autoreactive T cell responses.
This study aimed to investigate the association of lipoprotein and triglyceride levels with all-cause mortality in a population free from diabetes and cardiovascular disease (CVD) at baseline. The European Guidelines on cardiovascular disease prevention state that in general total cholesterol (TC) should be < 5 mmol/L (190 mg/dL) and low-density lipoprotein cholesterol (LDL-C) should be < 3 mmol/L (115 mg/dL).
A population-based register study in the period 1999–2007 including 118 160 subjects aged 50 + without statin use at baseline. All-cause mortality was related to lipoprotein and triglyceride levels and adjusted for statin use after inclusion.
All-cause mortality was lower in the groups with TC or LDL-C above the recommended levels. Compared with subjects with TC < 5 mmol/L, adjusted hazard ratios for the group aged 60–70 years ranged from 0.68 (95% confidence interval (CI) 0.61–0.77) for TC 5–5.99 mmol/L to 0.67 (95% CI 0.59–0.75) for TC 6–7.99 mmol/L and 1.02 (95% CI 0.68–1.53) for TC ≥ 8 mmol/L in males and from 0.57 (95% CI 0.48–0.67) to 0.59 (95% CI 0.50–0.68) and 1.02 (95% CI: 0.77–1.37) in females. For triglycerides, ratios compared with the group < 1 mmol/L in the females aged 60–70 years ranged from 1.04 (95% CI 0.88–1.23) to 1.35 (95% CI 1.10–1.66) and 1.25 (95% CI 1.05–1.48) for triglycerides 1–1.39 mmol/L, 1.4–1.69 mmol/L, and ≥ 1.7 mmol/L, respectively. Statin treatment after inclusion provided a survival benefit.
These associations indicate that high lipoprotein levels do not seem to be definitely harmful in the general population. However, high triglyceride levels in females are associated with decreased survival.
Cholesterol; Denmark; epidemiology; general practice; lipids; lipoproteins; mortality
To estimate the risk of spontaneous abortion after use of antidepressant medication during pregnancy.
From the Danish Medical Birth Registry and the Danish National Hospital Registry, we identified all pregnancies leading to in- or outpatient contacts in Denmark from February 1997 to December 2008. The Danish Registry of Medicinal Product Statistics provided information on the women's prescriptions for antidepressants during pregnancy. We obtained information on women who were diagnosed with depression from the Danish Psychiatric Central Registry. Adjusted relative risks (aRR) of spontaneous abortion were estimated according to exposure to antidepressants or maternal depression using binomial regression.
Of the 1,005,319 pregnancies (547,300 women) identified, 114,721 (11.4%) ended in a spontaneous abortion. We identified 22,061 pregnancies exposed to antidepressants and 1,843 with a diagnosis of depression with no antidepressant use, of which 2,637 (12.0%) and 205 (11.1%) ended in a spontaneous abortion, respectively. Antidepressant exposure was associated with an aRR of 1.14 (95% confidence interval (CI) 1.10–1.18) for spontaneous abortion compared with no exposure to antidepressants. Among women with a diagnosis of depression, the aRR for spontaneous abortion after any antidepressant exposure was 1.00 (95% CI 0.80–1.24). No individual selective serotonin reuptake inhibitor (SSRI) was associated with spontaneous abortions. In unadjusted analyses, we found that mirtazapine, venlafaxine, and duloxetine were associated with spontaneous abortions among women with depression but we had no information on potential differences in disease severity and only few pregnancies were exposed in the population.
We identified a slightly increased risk of spontaneous abortion associated with the use of antidepressants during pregnancy. However, among women with a diagnosis of depression, antidepressants in general or individual SSRI in particular were not associated with spontaneous abortions. Further studies are warranted on the newer non-SSRI antidepressants, as we had insufficient data to adjust for important confounding factors.
Drug-related adverse events remain an important cause of morbidity and mortality and impose huge burden on healthcare costs. Routinely collected electronic healthcare data give a good snapshot of how drugs are being used in ‘real-world’ settings.
To describe a strategy that identifies potentially drug-induced acute myocardial infarction (AMI) from a large international healthcare data network.
Post-marketing safety surveillance was conducted in seven population-based healthcare databases in three countries (Denmark, Italy, and the Netherlands) using anonymised demographic, clinical, and prescription/dispensing data representing 21,171,291 individuals with 154,474,063 person-years of follow-up in the period 1996–2010. Primary care physicians’ medical records and administrative claims containing reimbursements for filled prescriptions, laboratory tests, and hospitalisations were evaluated using a three-tier triage system of detection, filtering, and substantiation that generated a list of drugs potentially associated with AMI. Outcome of interest was statistically significant increased risk of AMI during drug exposure that has not been previously described in current literature and is biologically plausible.
Overall, 163 drugs were identified to be associated with increased risk of AMI during preliminary screening. Of these, 124 drugs were eliminated after adjustment for possible bias and confounding. With subsequent application of criteria for novelty and biological plausibility, association with AMI remained for nine drugs (‘prime suspects’): azithromycin; erythromycin; roxithromycin; metoclopramide; cisapride; domperidone; betamethasone; fluconazole; and megestrol acetate.
Although global health status, co-morbidities, and time-invariant factors were adjusted for, residual confounding cannot be ruled out.
A strategy to identify potentially drug-induced AMI from electronic healthcare data has been proposed that takes into account not only statistical association, but also public health relevance, novelty, and biological plausibility. Although this strategy needs to be further evaluated using other healthcare data sources, the list of ‘prime suspects’ makes a good starting point for further clinical, laboratory, and epidemiologic investigation.
Heparan sulfates (HSs) have potential therapeutic value as anti-inflammatory and antimetastasis drugs, in addition to their current use as anticoagulants. Recent advances in chemoenzymatic synthesis of HS provide a way to conveniently produce homogenous HS with different biological properties. Crystal structures of sulfotransferases involved in this process are providing atomic detail of their substrate binding clefts and interactions with their HS substrates. In theory, the flexibility of this method can be increased by modifying the specificities of the sulfotransferases based on the structures, thereby producing a new array of products.
To evaluate positive predictive value (PPV) of different disease codes and free text in identifying acute myocardial infarction (AMI) from electronic healthcare records (EHRs).
Validation study of cases of AMI identified from general practitioner records and hospital discharge diagnoses using free text and codes from the International Classification of Primary Care (ICPC), International Classification of Diseases 9th revision-clinical modification (ICD9-CM) and ICD-10th revision (ICD-10).
Population-based databases comprising routinely collected data from primary care in Italy and the Netherlands and from secondary care in Denmark from 1996 to 2009.
A total of 4 034 232 individuals with 22 428 883 person-years of follow-up contributed to the data, from which 42 774 potential AMI cases were identified. A random sample of 800 cases was subsequently obtained for validation.
Main outcome measures
PPVs were calculated overall and for each code/free text. ‘Best-case scenario’ and ‘worst-case scenario’ PPVs were calculated, the latter taking into account non-retrievable/non-assessable cases. We further assessed the effects of AMI misclassification on estimates of risk during drug exposure.
Records of 748 cases (93.5% of sample) were retrieved. ICD-10 codes had a ‘best-case scenario’ PPV of 100% while ICD9-CM codes had a PPV of 96.6% (95% CI 93.2% to 99.9%). ICPC codes had a ‘best-case scenario’ PPV of 75% (95% CI 67.4% to 82.6%) and free text had PPV ranging from 20% to 60%. Corresponding PPVs in the ‘worst-case scenario’ all decreased. Use of codes with lower PPV generally resulted in small changes in AMI risk during drug exposure, but codes with higher PPV resulted in attenuation of risk for positive associations.
ICD9-CM and ICD-10 codes have good PPV in identifying AMI from EHRs; strategies are necessary to further optimise utility of ICPC codes and free-text search. Use of specific AMI disease codes in estimation of risk during drug exposure may lead to small but significant changes and at the expense of decreased precision.
Epidemiology; Statistics & Research Methods
In a large population-based cohort in Denmark to examine if maternal use of antibiotics during pregnancy, as a marker of infection, increases the risk of febrile seizures in childhood in a large population-based cohort in Denmark.
All live-born singletons born in Denmark between January 1, 1996 and September 25, 2004 and who were alive on the 90th day of life were identified from the Danish National Birth Registry. Diagnoses of febrile seizures were obtained from the Danish National Hospital Register and maternal use of antibiotics was obtained from the National Register of Medicinal Product Statistics. Hazard ratios (HR) and 95% confidence intervals (95% CI) were estimated by Cox proportional hazard regression models.
We followed 551,518 singletons for up to 5 years and identified a total of 21,779 children with a diagnosis of febrile seizures. Slightly increased hazard ratios were observed among most exposure groups when compared to the unexposed group, ex. HR 1.08 95% CI: 1.05–1.11 for use of any systemic antibiotic during pregnancy.
We found weak associations between the use of pharmacologically different antibiotics during pregnancy and febrile seizures in early childhood which may indicate that some infections, or causes or effects of infections, during pregnancy could affect the fetal brain and induce susceptibility to febrile seizures.
Background: Diarylheptanoid (D3) isolated from the medicinal plant, Curcuma comosa, has estrogenic activity.
Objective: We aimed to elucidate the mechanism(s) of D3 action and compare it with that of 17β-estradiol (E2) using both in vitro and in vivo uterine models.
Methods: We used human uterine (Ishikawa) cells to determine the estrogenic action of D3 on the activation and nuclear translocation of estrogen receptor α (ERα). In addition, we further characterized the uterine response to D3 treatment in vivo.
Results: D3 activated an estrogen responsive element (ERE) luciferase reporter through ERα, and molecular modeling suggested that D3 could be accommodated in the ERα binding pocket. Using modified ERα to assay ligand-dependent nuclear translocation, we observed D3-dependent ERα interaction and translocation. In mouse uteri, early- and late-phase estrogen-regulated gene responses were increased in D3-treated ovariectomized wild-type animals, in a manner similar to that of E2; no response was seen in ERα knockout animals. We observed a divergence in estrogen responses after D3 treatment: D3 induced robust DNA synthesis in uterine epithelial cells, linked to an increase in cell-cycle–related genes; however, no increase in uterine weight was observed 24 hr after treatment. D3 also affected uterine progesterone receptor expression patterns similar to E2. When D3 and E2 were administered together, we observed no additive or antagonistic effects of D3 on E2. Our findings suggest that D3 is a weak estrogenic agonist compound.
Conclusion: D3 is a weakly acting phytoestrogen that mimics the mitogenic responses produced by E2 in an ERα-dependent manner, but it is unable to increase uterine weight or enhance or antagonize the effects of estrogen.
diarylheptanoid; ER-dependent; nuclear translocation; phytoestrogen; uterus
We have developed a fluorescence-based fiber-optical biosensor, which can selectively detect different antibodies in serial at preselected positions inside a single piece of fiber. The fiber is a microstructured polymer optical fiber fabricated from TOPAS cyclic olefin copolymer, which allows for UV activation of localized sensor layers inside the holes of the fiber. Serial fluorescence-based selective sensing of Cy3-labelled α-streptavidin and Cy5-labelled α-CRP antibodies is demonstrated.
microstructured polymer optical fiber; fluorescence; antibodies
Background and Aim
Maternal infections during pregnancy have been associated with several neurological disorders in the offspring. However, given the lack of specificity for both the exposures and the outcomes, other factors related to infection such as impaired maternal immune function may be involved in the causal pathway. If impaired maternal immune function plays a role, we would expect infection before pregnancy to be associated with these neurological outcomes.
The study population included all first-born singletons in Denmark between January 1 1982 and December 31 2004. We identified women who had hospital-recorded infections within the 5 year period before pregnancy, and women who had hospital-recorded infections during pregnancy. We grouped infections into either infections of the genitourinary system, or any other infections. Cox models were used to estimate adjusted hazard ratios (aHRs) with 95% confidence interval (CI). Maternal infection of the genitourinary system during pregnancy was associated with an increased risk of cerebral palsy (aHR = 1.63, 95% CI: 1.34–1.98) and epilepsy (aHR = 1.27, 95% CI: 1.13–1.42) in the children, compared to children of women without infections during pregnancy. Among women without hospital-recorded infections during pregnancy, maternal infection before pregnancy was associated with an increased risk of epilepsy (aHR = 1.35, 95% CI: 1.21–1.50 for infections of the genitourinary system, and HR = 1.12, 95% CI: 1.03–1.22 for any other infections) and a slightly higher risk of cerebral palsy (aHR = 1.20, 95% CI: 0.96–1.49 for infections of the genitourinary system, and HR = 1.23, 95% CI: 1.06–1.43 for any other infections) in the children, compared to children of women without infections before (and during) pregnancy.
These findings indicate that the maternal immune system, maternal infections, or factors related to maternal immune function play a role in the observed associations between maternal infections before pregnancy and cerebral diseases in the offspring.
AIM: To develop and validate a case definition of eosinophilic esophagitis (EoE) in the linked Danish health registries.
METHODS: For case definition development, we queried the Danish medical registries from 2006-2007 to identify candidate cases of EoE in Northern Denmark. All International Classification of Diseases-10 (ICD-10) and prescription codes were obtained, and archived pathology slides were obtained and re-reviewed to determine case status. We used an iterative process to select inclusion/exclusion codes, refine the case definition, and optimize sensitivity and specificity. We then re-queried the registries from 2008-2009 to yield a validation set. The case definition algorithm was applied, and sensitivity and specificity were calculated.
RESULTS: Of the 51 and 49 candidate cases identified in both the development and validation sets, 21 and 24 had EoE, respectively. Characteristics of EoE cases in the development set [mean age 35 years; 76% male; 86% dysphagia; 103 eosinophils per high-power field (eos/hpf)] were similar to those in the validation set (mean age 42 years; 83% male; 67% dysphagia; 77 eos/hpf). Re-review of archived slides confirmed that the pathology coding for esophageal eosinophilia was correct in greater than 90% of cases. Two registry-based case algorithms based on pathology, ICD-10, and pharmacy codes were successfully generated in the development set, one that was sensitive (90%) and one that was specific (97%). When these algorithms were applied to the validation set, they remained sensitive (88%) and specific (96%).
CONCLUSION: Two registry-based definitions, one highly sensitive and one highly specific, were developed and validated for the linked Danish national health databases, making future population-based studies feasible.
Eosinophilic esophagitis; Denmark; Epidemiology; Case definition; Sensitivity; Specificity
It is necessary to develop efficient methods to produce renewable fuels from lignocellulosic biomass. One of the main challenges to the industrialization of lignocellulose conversion processes is the large amount of cellulase enzymes used for the hydrolysis of cellulose. One method for decreasing the amount of enzyme used is to recycle the enzymes. In this study, the recycle of enzymes associated with the insoluble solid fraction after the enzymatic hydrolysis of cellulose was investigated for pretreated corn stover under a variety of recycling conditions.
It was found that a significant amount of cellulase activity could be recovered by recycling the insoluble biomass fraction, and the enzyme dosage could be decreased by 30% to achieve the same glucose yields under the most favorable conditions. Enzyme productivity (g glucose produced/g enzyme applied) increased between 30 and 50% by the recycling, depending on the reaction conditions. While increasing the amount of solids recycled increased process performance, the methods applicability was limited by its positive correlation with increasing total solids concentrations, reaction volumes, and lignin content of the insoluble residue. However, increasing amounts of lignin rich residue during the recycle did not negatively impact glucose yields.
To take advantage of this effect, the amount of solids recycled should be maximized, based on a given processes ability to deal with higher solids concentrations and volumes. Recycling of enzymes by recycling the insoluble solids fraction was thus shown to be an effective method to decrease enzyme usage, and research should be continued for its industrial application.
Glucose-6-phosphate is imported into the amyloplast of potato tubers and thought to constitute the precursor for starch synthesis in potato tubers. However, recently it was shown that glucose-1-phosphate can also be imported into the amyloplast and incorporated into starch via an ATP independent mechanism under special conditions. Nonetheless, glucose-6-phosphate is believed to be the quantitatively important precursor for starch synthesis in potato.
Potato tubers of the high yielding cv Kuras had low gene expression of plastidial phophoglucomutase (PGM) and normal levels of transcripts for other enzymes involved in starch metabolism in comparison with medium and low yielding cultivars as determined by DeepSAGE transcriptome profiling. The decrease in PGM activity in Kuras was confirmed by measuring the enzyme activity from potato tuber extracts. Contrary to expectations, this combination lead to a higher level of intracellular glucose-1-phosphate (G1P) in Kuras suggesting that G1P is directly imported into plastids and can be quantitatively important for starch synthesis under normal conditions in high yielding cultivars.
This could open entirely new possibilities for metabolic engineering of the starch metabolism in potato via the so far uncharacterized G1P transporter. The perspectives are to increase yield and space efficiency of this important crop. In the light of the increasing demands imposed on agriculture to support a growing global population this presents an exciting new possibility.
Earlier studies suggest a protective association between vitamin K antagonist (VKA) anticoagulants and the incidence of cancer. The authors examined the associations between VKA therapy and incidence of 24 site-specific cancers with a Danish population-based cohort study, using heart valve replacement as an instrumental variable. The authors enrolled 9,727 Danish residents who received a replacement heart valve between 1989 and 2006. The heart valve recipients were matched with 95,481 unexposed individuals on age and sex. The authors used the heart valve replacement instrument to estimate rate ratios associating VKA therapy with incidence of the 24 site-specific cancers using Poisson regression models. Direct associations between VKA therapy and incidence of the 24 cancers were estimated in a prescription validation subset. The instrumental variable associations were plotted according to the inverse normal of rank percentile and subjected to semi-Bayes shrinkage adjustment for multiple comparisons. The pattern of associations was consistent with a null-centered Gaussian distribution. No individual cancer site showed a substantial positive or negative association with VKA therapy in the prescription validation subset, the instrumental variable analysis, or the analysis with semi-Bayes adjustment. These results do not support the existing hypothesis that VKA therapy is associated with reduced cancer risk.
anticoagulants; bias (epidemiology); heart valves; instrumental variable; neoplasms; vitamin K
The Danish health care system provides partial reimbursement of most prescription medications in Denmark. The dispensation of prescription medications is registered in administrative databases. Each time a prescription is redeemed at a pharmacy, an electronic record is generated with information related to the user, prescriber, the pharmacy, and the dispensed drug. The National Health Service gathers this information for administration of the drug reimbursement plan. Recently, this information became the basis for the establishment of a new research database, the Danish National Database of Reimbursed Prescriptions (DNDRP). In this paper, we review the content, coverage, quality, linkage, access, and research possibilities of this new database. The database encompasses the reimbursement records of all reimbursed drugs sold in community pharmacies and hospital-based outpatient pharmacies in Denmark since 2004. On average, approximately 3.5 million users are recorded in the database each year. During the coverage period, the number of annual prescription redemptions increased by 15%. Most dispensed prescriptions are in the categories “alimentary tract and metabolism”, “cardiovascular system”, “nervous system”, and “respiratory system”. Individuals are identified by the unique central personal registration (CPR) number assigned to all persons born in or immigrating to Denmark. The new database fully complies with Denmark’s Act on Processing of Personal Data, while avoiding additional restrictions imposed on data use at the Danish National Prescription Registry, administered by Statistics Denmark. Most importantly, CPR numbers are reversibly encrypted, which allows re-identification of drug users; furthermore, the data access is possible outside the servers of Statistics Denmark. These features open additional opportunities for international collaboration, validation studies, studies on adverse drug effects requiring review of medical records, studies involving contact to general practitioners, and linkage of prescription data to other clinical and research databases. The DNDRP thus is a valuable data source for pharmacoepidemiological research.
Denmark; patient registration; pharmacoepidemiology; registry-based research
Accumulating evidence suggests that statins affect diseases other than cardiovascular disease, including cancer, and that these effects may depend on the lipid solubility of specific statins. Though many studies have reported an association between statin use and breast cancer incidence, the relationship between statin use and breast cancer recurrence has not been well studied.
We conducted a nationwide, population-based prospective cohort study of all female residents in Denmark diagnosed with stage I–III invasive breast carcinoma who were reported to the Danish Breast Cancer Cooperative Group registry between 1996 and 2003 (n = 18 769). Women were followed for a median of 6.8 years after diagnosis. Prescriptions for lipophilic and hydrophilic statins were ascertained from the national electronic pharmacy database. Associations between statin prescriptions and breast cancer recurrence were estimated with generalized linear models and Cox proportional hazards regression with adjustment for age and menopausal status at diagnosis; histological grade; estrogen receptor status; receipt of adjuvant therapy; type of primary surgery received; pre-diagnosis hormone replacement therapy; and co-prescriptions of aspirin, angiotensin-converting enzyme inhibitors, nonsteroidal anti-inflammatory drugs, or anticoagulants. All statistical tests were two-sided.
Most prescriptions for lipophilic statins in the study population were for simvastatin. Exclusive simvastatin users experienced approximately 10 fewer breast cancer recurrences per 100 women after 10 years of follow-up (adjusted 10-year risk difference = −0.10, 95% confidence interval = −0.11 to −0.08), compared with women who were not prescribed a statin. Exclusive hydrophilic statin users had approximately the same risk of breast cancer recurrence as women not prescribed a statin over follow-up (adjusted 10-year risk difference = 0.05, 95% confidence interval = −0.01 to 0.11).
Simvastatin, a highly lipophilic statin, was associated with a reduced risk of breast cancer recurrence among Danish women diagnosed with stage I–III breast carcinoma, whereas no association between hydrophilic statin use and breast cancer recurrence was observed.
This paper provides an overview of the baseline data collected in the nationwide Danish Centre for Strategic Research in Type 2 Diabetes (DD2) project. The paper presents descriptive data from the first 580 patients enrolled in the DD2. The DD2 database will contain detailed interview data, clinical examination data, and urine and blood samples from up to 10,000 patients newly diagnosed with type 2 diabetes each year, collected from general practitioners and hospital outpatient clinics in all of Denmark. Of the first DD2 patients enrolled, blood and urine samples have been obtained from 97%. The median age of the first 580 patients was 59 years and 322 (56%) were men. Median weight gain from age 20 to maximum lifetime weight was 29 kg for men and 31 kg for women, and 364 patients (63%) did not currently participate in regular sports activities. Two hundred and ninety two patients (50%) had a known family history of diabetes. Two hundred fifty (43%) of the 580 DD2 patients have also been enrolled in the Danish Diabetes Database for Adults from which additional clinical data can be obtained. Among these 250 patients (154 of whom were men, 96 women), 75 (49%) men were currently obese, and 63 (41%) were overweight, whereas 62 (65%) women were obese, and another 21 (22%) were overweight. Twenty-nine patients (12%) received insulin, 164 patients (66%) received oral antidiabetics only, and 57 (23%) received no antidiabetic treatment. Glycemic regulation was modest (the glycosylated hemoglobin A of 46% was ≥7.5%). Two thirds of the patients received antihypertensive and hypolipidemic treatment. Self-reported daily tobacco smoking (23%) and alcohol overuse (6%) seemed comparable to occurrence in the general Danish population. One quarter of the patients with newly diagnosed diabetes had a history of hospital-diagnosed comorbidity at baseline as included in the Charlson comorbidity index, in particular prior myocardial infarction (5%), cerebrovascular disease (5%), peripheral vascular disease (4%), chronic pulmonary disease (6%), and previous solid cancer (6%). In the future, the DD2 database represents a valuable source for outcome studies in type 2 diabetes.
type 2 diabetes; epidemiological methods; registries; prognosis
Postpartum haemorrhage (PPH) remains a leading cause of maternal mortality worldwide. In Denmark 2% of parturients receive blood transfusion. During the course of bleeding fibrinogen (coagulation factor I) may be depleted and fall to critically low levels, impairing haemostasis and thus worsening the ongoing bleeding. A plasma level of fibrinogen below 2 g/L in the early phase of postpartum haemorrhage is associated with subsequent development of severe haemorrhage. Use of fibrinogen concentrate allows high-dose substitution without the need for blood type crossmatch. So far no publications of randomised controlled trials involving acutely bleeding patients in the obstetrical setting have been published. This trial aims to investigate if early treatment with fibrinogen concentrate reduces the need for blood transfusion in women suffering severe PPH.
In this randomised placebo-controlled double-blind multicentre trial, parturients with primary PPH are eligible following vaginal delivery in case of: manual removal of placenta (blood loss ≥ 500 ml) or manual exploration of the uterus after the birth of placenta (blood loss ≥ 1000 ml). Caesarean sections are also eligible in case of perioperative blood loss ≥ 1000 ml. The exclusion criteria are known inherited haemostatic deficiencies, prepartum treatment with antithrombotics, pre-pregnancy weight <45 kg or refusal to receive blood transfusion. Following informed consent, patients are randomly allocated to either early treatment with 2 g fibrinogen concentrate or 100 ml isotonic saline (placebo). Haemostatic monitoring with standard laboratory coagulation tests and thromboelastography (TEG, functional fibrinogen and Rapid TEG) is performed during the initial 24 hours.
Primary outcome is the need for blood transfusion. To investigate a 33% reduction in the need for blood transfusion, a total of 245 patients will be included. Four university-affiliated public tertiary care hospitals will include patients during a two-year period. Adverse events including thrombosis are assessed in accordance with International Conference on Harmonisation (ICH) good clinical practice (GCP).
A widespread belief in the benefits of early fibrinogen substitution in cases of PPH has led to increased off-label use. The FIB-PPH trial is investigator-initiated and aims to provide an evidence-based platform for the recommendations of the early use of fibrinogen concentrate in PPH.
Postpartum haemorrhage; Haemostasis; Blood transfusion; Fibrinogen concentrate; Obstetrics; Thrombelastography; Coagulation
Peanut allergy affects 1% of the population and causes the most fatal food-related anaphylactic reactions. The protein Ara h 2 is the most potent peanut allergen recognized by 80–90% of peanut allergic patients.
The crystal structure of the major peanut allergen Ara h 2 was determined for the first time at 2.7 Å resolution using a customized MBP-fusion system. IgE antibody binding to the MBP fusion construct versus the natural allergen was compared by ELISA using sera from peanut allergic patients.
The structure of Ara h 2 is a five helix bundle held together by four disulfide bonds and related to the prolamin protein superfamily. The fold is most similar to other amylase and trypsin inhibitors. The MBP-Ara h 2 fusion construct was positively recognized by IgE from 76% of allergic patients (25/33). Two populations of patients could be identified. Sub-population 1 (n=14) showed an excellent correlation of IgE antibody binding to natural versus recombinant Ara h 2. Sub-population 2 (n=15) showed significantly reduced IgE binding to the MBP fusion protein. Interestingly, about 20% of the IgE binding in sub-population 2 could be recovered by increasing the distance between MBP and Ara h 2 in a second construct.
The reduced IgE binding to the MBP-Ara h 2 of sub-population 2 indicates that the MBP molecule protects an immunodominant epitope region near the first helix of Ara h 2. Residues involved in the epitope(s) are suggested by the crystal structure. The MBP-Ara h 2 fusion constructs will be useful to further elucidate the relevance of certain epitopes to peanut allergy.
Peanut; Allergy; Ara h 2; Immunotherapy; Structure
Although most DNA polymerases discriminate against ribonucleotide triphosphaets (rNTPs) during DNA synthesis, recent studies have shown that large numbers of ribonucleotides are incorporated into the eukaryotic nuclear genome. Here, we investigate how a DNA polymerase can stably incorporate an rNTP. The X-ray crystal structure of a variant of human DNA polymerase λ reveals that the rNTP occupies the nucleotide binding pocket without distortion of the active site, despite an unfavorable interaction between the 2′-O and Tyr505 backbone carbonyl. This indicates an energetically unstable binding state for the rNTP, stabilized by additional protein–nucleotide interactions. Supporting this idea is the 200-fold lower catalytic efficiency for rNTP relative to deoxyribonucleotide triphosphate (dNTP) incorporation, reflecting a higher apparent Km value for the rNTP. Furthermore, distortion observed in the structure of the post-catalytic product complex suggests that once the bond between the α- and β-phosphates of the rNTP is broken, the unfavorable binding state of the ribonucleotide cannot be maintained. Finally, structural and biochemical evaluation of dNTP insertion onto an ribonucleotide monophosphate (rNMP)-terminated primer indicates that a primer-terminal rNMP does not impede extension. The results are relevant to how ribonucleotides are incorporated into DNA in vivo, during replication and during repair, perhaps especially in non-proliferating cells when rNTP:dNTP ratios are high.
Mismatch repair (MMR) corrects replication errors that would otherwise lead to mutations and, potentially, various forms of cancer. Among several proteins required for eukaryotic MMR, MutLα is a heterodimer comprised of Mlh1 and Pms1. The two proteins dimerize along their C-terminal domains (CTDs), and the CTD of Pms1 houses a latent endonuclease that is required for MMR. The highly-conserved N-terminal domains (NTDs) independently bind DNA and possess ATPase active sites. Here we use two protein footprinting techniques, limited proteolysis and oxidative surface mapping, coupled with mass spectrometry to identify amino acids involved along the DNA-binding surface of the Pms1-NTD. Limited proteolysis experiments elucidated several basic residues that were protected in the presence of DNA, while oxidative surface mapping revealed one residue that is uniquely protected from oxidation. Furthermore, additional amino acids distributed throughout the Pms1-NTD were protected from oxidation either in the presence of a non-hydrolyzable analog of ATP or DNA, indicating that each ligand stabilizes the protein in a similar conformation. Based on the recently published X-ray crystal structure of yeast Pms1-NTD, a model of the Pms1-NTD/DNA complex was generated using the mass spectrometric data as constraints. The proposed model defines the DNA-binding interface along a positively-charged groove of the Pms1-NTD and complements prior mutagenesis studies of E. coli and eukaryotic MutL.
Mismatch repair; MutLα; Pms1; DNA binding; mass spectrometry
Cytochrome P450 2D6 (CYP2D6) inhibition reduces the concentration of 4-hydroxylated tamoxifen metabolites, but the clinical relevance remains uncertain.
We conducted a large case–control study nested in the population of 11 251 women aged 35–69 years at diagnosis of stage I–III breast cancer between 1985 and 2001 on Denmark’s Jutland Peninsula and registered with the Danish Breast Cancer Cooperative Group. We identified 541 recurrent or contralateral breast cancers among women with estrogen receptor–positive (ER+) disease treated with tamoxifen for at least 1 year and 300 cancers in women with ER-negative (ER−) disease never treated with tamoxifen. We matched one control subject per case patient on ER status, menopausal status, stage, calendar time, and county, genotyped the CYP2D6*4 allele to assess genetic inhibition, and ascertained prescription history to assess drug–drug inhibition. We estimated the odds ratio (OR), associating CYP2D6 inhibition with breast cancer recurrence and adjusted for potential confounding with logistic regression. To address bias from incomplete information on CYP2D6 function, we used Monte Carlo simulation to complete a record-level probabilistic bias analysis. All statistical tests were two-sided.
The frequency of the CYP2D6*4 minor allele was 24% in case patients with ER+ tumors, 23% in case patients with ER− tumors, and 22% each in control subjects with ER+ and ER− tumors. In women with ER+ tumors, the associations of one functional allele with recurrence (OR = 0.99; 95% confidence interval = 0.76 to 1.3) and no functional allele with recurrence (OR = 1.4; 95% confidence interval = 0.84 to 2.3) were near null, as were those for women with ER− tumors. The near-null associations persisted when evaluated by intake of medications, by combining genotype with medication history, in the probabilistic bias analysis, or by restricting the analysis to women with ER expression confirmed by re-assay.
The association between CYP2D6 inhibition and recurrence in tamoxifen-treated patients is likely null or small.
Glucocorticoids are widely prescribed drugs. In the human body, glucocorticoid is the main stress hormone and controls a variety of physiological and cellular processes, including metabolism and immune response. It belongs to the same steroid superfamily as estrogens, which are known to play a role in breast cancer. However, the effect of glucocorticoid use on the risk of breast cancer is not clear.
We conducted a case-control study using population-based medical databases from Northern Denmark (1.8 million inhabitants) to investigate the association between glucocorticoid prescriptions and breast cancer risk. The study included 9,488 incident breast cancer cases diagnosed between 1994 and 2008 and 94,876 population controls. We estimated the odds ratios (ORs) and 95% confidence intervals (CIs) associating glucocorticoid use with breast cancer occurrence, controlling for prescriptions of postmenopausal hormone replacement therapy, anti-diabetics, immunosuppressive drugs, and hospital diagnosis of obesity, diabetes, chronic pulmonary diseases and autoimmune diseases.
We found no effect on breast cancer risk in ever users (> 2 prescriptions) of any glucocorticoids (adjusted odds ratio (aOR) = 1.0; 95% CI: 0.96, 1.1), systemic glucocorticoids (aOR = 1.0; 95% CI: 0.96, 1.1), or inhaled glucocorticoids (aOR = 1.0; 95% CI: 0.95, 1.1), each compared to never users of any glucocorticoids. Associations for recent use (preceding two years) and former use (more than two years earlier) were near null in all dose categories (low, medium and high number of prescriptions). Intensity of systemic glucocorticoid use (cumulative prednisolone equivalent doses), regardless of duration (< 1, 1 to 5, 5+ years), was also not associated with breast cancer risk.
Overall, our study provides no evidence that glucocorticoid use affects the risk of breast cancer.